JPWO2020257356A5 - - Google Patents

Description

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are specifically and individually indicated as if each individual publication, patent, or patent application was incorporated by reference. incorporated herein by reference to the same extent.
[Invention 1001]
a) an amplification chamber fluidly connected to the valve;
b) a detection chamber fluidly connected to a valve connected to the sample metering channel;
c) a detection reagent chamber fluidly connected to said detection chamber via a resistance channel, said detection reagent chamber containing a programmable nuclease, a guide nucleic acid and a labeled detector nucleic acid, said labeled detector nucleic acid being linked to a segment of a target nucleic acid; said detection reagent chamber that can be cleaved by binding of said guide nucleic acid of
A microfluidic cartridge for detecting a target nucleic acid comprising:
[Invention 1002]
1002. The microfluidic cartridge of invention 1001, wherein said sample metering channel controls the volume of liquid dispensed within the channel or chamber.
[Invention 1003]
1003. The microfluidic cartridge of present invention 1002, wherein said sample metering channel is fluidly connected to said detection chamber.
[Invention 1004]
1004. The resistance channel of any of inventions 1001-1003, wherein said resistance channel has a serpentine path, an angled path, or a circuitous path.
[Invention 1005]
1004. The microfluidic cartridge of any of the inventions 1001-1004, wherein said valve is a rotary valve, pneumatic valve, hydraulic valve, elastomeric valve.
[Invention 1006]
1006. The microfluidic cartridge of any of the inventions 1001-1005, wherein said resistance channel is fluidly connected with said valve.
[Invention 1007]
1006. The microfluidic cartridge of any of inventions 1001-1006, wherein said valve comprises a casing comprising a "substrate" or "overmold".
[Invention 1008]
1008. The microfluidic cartridge of any of the inventions 1001-1007, wherein said valve is actuated by a solenoid.
[Invention 1009]
wherein the valve is manually, magnetically, electrically, thermally, by a bistable circuit, by piezoelectric material, electrochemically, by phase change, rheologically, pneumatically, by a non-return valve, The microfluidic cartridge of any of the inventions 1001-1008 controlled by capillary action or any combination thereof.
[Invention 1010]
1009. The microfluidic cartridge of any of inventions 1005-1009, wherein said rotary valve fluidly connects at least 3, at least 4, or at least 5 chambers.
[Invention 1011]
1011. The microfluidic cartridge of any of inventions 1001-1010, further comprising an amplification reagent chamber fluidly connected to said amplification chamber.
[Invention 1012]
1012. The microfluidic cartridge of the present invention 1011, further comprising a sample chamber fluidly connected to said amplification reagent chamber.
[Invention 1013]
1013. The microfluidic cartridge of the present invention 1012, further comprising a sample inlet connected to said sample chamber.
[Invention 1014]
1013. The microfluidic cartridge of Invention 1013, wherein said sample inlet is sealable.
[Invention 1015]
1015. The microfluidic cartridge of Invention 1014, wherein said sample inlet forms a seal around a sample.
[Invention 1016]
The microfluidic cartridge of any of inventions 1012-1015, wherein said sample chamber contains a lysis buffer.
[Invention 1017]
The microfluidic cartridge of any of inventions 1012-1016, further comprising a lysis buffer storage chamber fluidly connected to said sample chamber.
[Invention 1018]
1017. The microfluidic cartridge of the present invention 1017, wherein said lysis buffer storage chamber contains a lysis buffer.
[Invention 1019]
The microfluidic cartridge of any of inventions 1016-1018, wherein said lysis buffer is a dual lysis/amplification buffer.
[Invention 1020]
The microfluidic cartridge of any of inventions 1017-1019, wherein said lysis buffer storage chamber is fluidly connected to said sample chamber via a second valve.
[Invention 1021]
1021. The microfluidic cartridge of any of the inventions 1012-1020, wherein said sample chamber is fluidly connected to said amplification chamber via said amplification reagent chamber.
[Invention 1022]
1021. The microfluidic cartridge of any of the inventions 1012-1020, wherein said sample chamber is fluidly connected to said amplification reagent chamber via said amplification chamber.
[Invention 1023]
1023. The microfluidic cartridge of any of the inventions 1011-1022, configured to direct fluid bi-directionally between said amplification reagent chamber and amplification chamber.
[Invention 1024]
1024. The microfluidic cartridge of any of inventions 1001-1023, wherein said detection reagent chamber is fluidly connected to said amplification chamber.
[Invention 1025]
1025. The microfluidic cartridge of any of inventions 1001-1024, wherein said amplification chamber is fluidly connected to said detection chamber via said detection reagent chamber.
[Invention 1026]
1026. The microfluidic cartridge of any of claims 1001-1025, further comprising a reagent port above said detection chamber, said reagent port configured to deliver fluid from said detection reagent chamber to said detection chamber. .
[Invention 1027]
1027. The microfluidic cartridge of any of inventions 1001-1026, wherein said amplification chamber is fluidly connected to said detection reagent chamber via said detection chamber.
[Invention 1028]
1028. The microfluidic cartridge of any of inventions 1001-1027, wherein said resistance channel is configured to reduce backflow into said detection chamber and into said detection reagent chamber.
[Invention 1029]
1028. The microfluidic cartridge of any of inventions 1002-1027, wherein the sample metering channel is configured to direct a volume of fluid from the detection reagent chamber to the detection chamber.
[Invention 1030]
1029. The microfluidic cartridge of any of inventions 1001-1029, wherein said amplification chamber and detection chamber are thermally isolated.
[Invention 1031]
1031. The microfluidic cartridge of any of the inventions 1001-1030, wherein said detection reagent chamber is fluidly connected to said detection chamber.
[Invention 1032]
1032. The microfluidic cartridge of any of inventions 1001-1031, wherein said detection reagent chamber is fluidly connected to said detection chamber via a second resistance channel.
[Invention 1033]
1033. The microfluidic cartridge of any of the inventions 1001-1032, wherein said resistance channel or said second resistance channel is a serpentine resistance channel.
[Invention 1034]
1033. The microfluidic cartridge of any of inventions 1001-1033, wherein said resistance channel or said second resistance channel comprises at least two hairpin portions.
[Invention 1035]
1034. The microfluidic cartridge of any of the inventions 1001-1034, wherein said resistance channel or said second resistance channel comprises at least 1, at least 2, at least 3, or at least 4 right angles.
[Invention 1036]
1035. The microfluidic cartridge of any of inventions 1001-1035, wherein said amplification chamber includes a sealable sample input port.
[Invention 1037]
1036. The microfluidic cartridge of invention 1036, wherein the sample input port is configured to form a seal around the swab.
[Invention 1038]
1038. The microfluidic cartridge of any of inventions 1001-1037, configured for connection to a first pump for pumping fluid from said amplification chamber to said detection chamber.
[Invention 1039]
1038. The microfluidic cartridge of any of the inventions 1001-1038, configured to be connected to a second pump for pumping fluid from said detection reagent chamber to said detection chamber.
[Invention 1040]
The microfluidic cartridge of any of Inventions 1038-1039, wherein the first pump or said second pump is a pneumatic, peristaltic, hydraulic, or syringe pump.
[Invention 1041]
The microfluidic cartridge of any of inventions 1001-1040, wherein said amplification chamber is fluidly connected to a port configured to receive air pressure.
[Invention 1042]
1041. The microfluidic cartridge of Invention 1041, wherein said amplification chamber is fluidly connected to said port via a channel.
[Invention 1043]
The microfluidic cartridge of any of inventions 1011-1042, wherein said amplification reagent chamber is connected to a second port configured to receive air pressure.
[Invention 1044]
1043. The microfluidic cartridge of Invention 1043, wherein said amplification reagent chamber is fluidly connected to said second port via a second channel.
[Invention 1045]
1044. The microfluidic cartridge of any of inventions 1011-1044, configured to be connected to a third pump for pumping fluid from said amplification reagent chamber to said amplification chamber.
[Invention 1046]
The microfluidic cartridge of Invention 1045, wherein said third pump is a pneumatic, peristaltic, hydraulic, or syringe pump.
[Invention 1047]
1046. The microfluidic cartridge of any of inventions 1001-1046, wherein said detection reagent chamber is connected to a port configured to receive air pressure.
[Invention 1048]
1048. The microfluidic cartridge of any of inventions 1001-1047, wherein said detection reagent chamber is fluidly connected to a third port via a third channel.
[Invention 1049]
1048. The microfluidic cartridge of any of inventions 1001-1048, configured to be connected to a fourth pump for pumping fluid from said detection reagent chamber to said detection chamber.
[Invention 1050]
The microfluidic cartridge of Invention 1049, wherein said fourth pump is a pneumatic, peristaltic, hydraulic, or syringe pump.
[Invention 1051]
1050. The microfluidic cartridge of any of inventions 1001-1050, further comprising a plurality of ports configured to couple to a gas manifold, said plurality of ports configured to receive air pressure.
[Invention 1052]
1052. The microfluidic cartridge of any of inventions 1001-1051, wherein any chamber of said microfluidic cartridge is connected to a plurality of ports of invention 1050.
[Invention 1053]
1053. The microfluidic cartridge of any of inventions 1001-1052, wherein said valve is opened by application of a current electrical signal.
[Invention 1054]
1053. The microfluidic cartridge of any of inventions 1001-1053, wherein said detection reagent chamber is circular.
[Invention 1055]
1053. The microfluidic cartridge of any of inventions 1001-1053, wherein said detection reagent chamber is elongated.
[Invention 1056]
1053. The microfluidic cartridge of any of inventions 1001-1053, wherein said detection reagent chamber is hexagonal.
[Invention 1057]
1057. The microfluidic cartridge of any of inventions 1002-1056, wherein the regions of said resistance channel are shaped to direct flow in a direction perpendicular to the net flow direction.
[Invention 1058]
1056. The microfluidic cartridge of any of claims 1002-1056, wherein regions of said resistance channel are shaped to direct flow in a direction perpendicular to an axis defined by two ends of said resistance channel.
[Invention 1059]
1058. The microfluidic cartridge of any of inventions 1002-1058, wherein regions of said resistance channel are shaped to direct flow along the z-axis of said microfluidic cartridge.
[Invention 1060]
1059. The microfluidic cartridge of any of inventions 1001-1059, wherein said valve is fluidly connected to two detection chambers via an amplification mixture splitter.
[Invention 1061]
1060. The microfluidic cartridge of any of inventions 1001-1060, wherein said valve is fluidly connected to 3, 4, 5, 6, 7, 8, 9, or 10 detection chambers via amplification mixture splitters.
[Invention 1062]
1061. The microfluidic cartridge of any of inventions 1001-1061, further comprising a second valve fluidly connected to said detection reagent chamber and to said detection chamber.
[Invention 1063]
1063. The microfluidic cartridge of any of inventions 1001-1062, wherein said detection chamber is vented with a hydrophobic PTFE vent.
[Invention 1064]
1063. The microfluidic cartridge of any of inventions 1001-1063, wherein said detection chamber comprises an optically transparent surface.
[Invention 1065]
1064. The microfluidic cartridge of any of inventions 1001-1064, wherein said amplification chamber is configured to hold between 10 μL and 500 μL of fluid.
[Invention 1066]
1065. The microfluidic cartridge of any of inventions 1011-1065, wherein said amplification reagent chamber is configured to hold between 10 μL and 500 μL of fluid.
[Invention 1067]
1066. The microfluidic cartridge of any of the inventions 1001-1066, configured to receive between 2 μL and 100 μL of sample containing nucleic acids.
[Invention 1068]
The microfluidic cartridge of any of inventions 1001-1067, wherein said amplification reagent chamber contains 5-200 μl of amplification buffer.
[Invention 1069]
1068. The microfluidic cartridge of any of inventions 1001-1068, wherein said amplification chamber contains 45 μl of amplification buffer.
[Invention 1070]
1069. The microfluidic cartridge of any of inventions 1001-1069, wherein said detection reagent chamber stores 5-200 μl of fluid containing said programmable nuclease, said guide nucleic acid and said labeled detector nucleic acid.
[Invention 1071]
The microfluidic cartridge of any of inventions 1001-1070 comprising 2, 3, 4, 5, 6, 7, or 8 detection chambers.
[Invention 1072]
The microfluidic cartridge of Invention 1071, wherein said 2, 3, 4, 5, 6, 7, or 8 detection chambers are fluidly connected to a single sample chamber.
[Invention 1073]
1073. The microfluidic cartridge of any of inventions 1001-1072, wherein said detection chamber holds up to 100 μL, 200 μL, 300 μL, or 400 μL of fluid.
[Invention 1074]
The microfluidic cartridge of any of Inventions 1001-1073 comprising 5-7 layers.
[Invention 1075]
The microfluidic cartridge of any of inventions 1001-1074 comprising the layers shown in Figure 130B.
[Invention 1076]
1075. The microfluidic cartridge of any of inventions 1001-1075, further comprising a sample input port configured to fit a slip luer tip.
[Invention 1077]
1076. The microfluidic cartridge of Invention 1076, wherein said slip luer tip is adapted to fit a sample-holding syringe.
[Invention 1078]
1078. The microfluidic cartridge of any of inventions 1076-1077, wherein said sample input port is hermetically sealable.
[Invention 1079]
The microfluidic cartridge of any of inventions 1001-1078, further comprising a slide valve.
[Invention 1080]
1079. The microfluidic cartridge of Invention 1079, wherein said slide valve connects said amplification reagent chamber to said amplification chamber.
[Invention 1081]
The microfluidic cartridge of invention 1079 or 1080, wherein said slide valve connects said amplification chamber to said detection reagent chamber.
[Invention 1082]
The microfluidic cartridge of any of inventions 1079-1081, wherein said slide valve connects said amplification reagent chamber to said detection chamber.
[Invention 1083]
A manifold configured to receive the microfluidic cartridge of any of the inventions 1001-1082.
[Invention 1084]
a pump configured to pump a fluid into the detection chamber; an illumination source configured to illuminate the detection chamber; and to detect a detectable signal produced by the labeled detector nucleic acid. 1083. The manifold of the present invention 1083, comprising a configured detector and a heater configured to heat said amplification chamber.
[Invention 1085]
1084. The manifold of present invention 1084, further comprising a second heater configured to heat said detection chamber.
[Invention 1086]
1086. The manifold of any of inventions 1084-1085, wherein said illumination source is a broad spectrum light source.
[Invention 1087]
The manifold of any of inventions 1084-1086, wherein the illumination source light provides illumination having a bandwidth of less than 5 nm.
[Invention 1088]
1087. The manifold of any of the present inventions 1084-1087, wherein said illumination source is a light emitting diode.
[Invention 1089]
The manifold of Invention 1088, wherein said light emitting diodes produce white, blue, or green light.
[Invention 1090]
1089. The manifold of any of claims 1084-1089, wherein said detectable signal is light.
[Invention 1091]
The manifold of any of the inventions 1084-1090, wherein said detector is a camera or a photodiode.
[Invention 1092]
The manifold of any of inventions 1084-1091, wherein said detector has a detection bandwidth of less than 100 nm, less than 75 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, or less than 5 nm.
[Invention 1093]
The manifold of any of inventions 1084-1092, further comprising an optical filter configured to reside between said detection chamber and said detector.
[Invention 1094]
1093. The microfluidic cartridge of any of inventions 1001-1093, wherein said amplification chamber contains amplification reagents.
[Invention 1095]
The microfluidic cartridge of any of Inventions 1011-1094, wherein said amplification reagent chamber contains amplification reagents.
[Invention 1096]
1095. The microfluidic cartridge of any of inventions 1094-1095, wherein said amplification reagents comprise primers, polymerase, dNTPs, amplification buffer.
[Invention 1097]
The microfluidic cartridge of any of inventions 1001-1096, wherein said amplification chamber contains a lysis buffer.
[Invention 1098]
The microfluidic cartridge of any of inventions 1011-1097, wherein said amplification reagent chamber contains a lysis buffer.
[Invention 1099]
The microfluidic cartridge of any of inventions 1094-1098, wherein said amplification reagent comprises reverse transcriptase.
[Invention 1100]
The microfluidic cartridge of any of Inventions 1094-1099, wherein said amplification reagents comprise reagents for thermocycling amplification.
[Invention 1101]
The microfluidic cartridge of any of Inventions 1094-1099, wherein said amplification reagents comprise reagents for isothermal amplification.
[Invention 1102]
The amplification reagent is transcription amplification (TMA), helicase dependent amplification (HDA), cyclic helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR) , rolling circle amplification (RCA), ligase chain reaction (LCR), simple methods for amplifying RNA targets (SMART), single-primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA) ), hinge-initiated primer-dependent nucleic acid amplification (HIP), nicking enzyme amplification reaction (NEAR), or modified multiple displacement amplification (IMDA).
[Invention 1103]
The microfluidic cartridge of any of inventions 1094-1102, wherein said amplification reagents comprise reagents for loop-mediated amplification (LAMP).
[Invention 1104]
The microfluidic cartridge of any of inventions 1016-1103, wherein said lysis buffer and said amplification buffer are a single buffer.
[Invention 1105]
The microfluidic cartridge of any of inventions 1016-1104, wherein said lysis buffer storage chamber contains a lysis buffer.
[Invention 1106]
1106. The microfluidic cartridge of any of the inventions 1016-1105, wherein said lysis buffer has a pH between pH 4 and pH 5.
[Invention 1107]
The microfluidic cartridge of any of inventions 1001-1106, further comprising a reverse transcription reagent.
[Invention 1108]
1107. The microfluidic cartridge of Invention 1107, wherein said reverse transcription reagents comprise reverse transcriptase, primers, and dNTPs.
[Invention 1109]
1108. The microfluidic cartridge of any of inventions 1001-1108, wherein said programmable nuclease comprises a RuvC catalytic domain.
[Invention 1110]
1109. The microfluidic cartridge of any of inventions 1001-1109, wherein said programmable nuclease is a type V CRISPR/Cas effector protein.
[Invention 1111]
1111. The microfluidic cartridge of invention 1110, wherein said type V CRISPR/Cas effector protein is a Casl2 protein.
[Invention 1112]
The present invention, wherein said Cas12 protein comprises a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide. 1111 microfluidic cartridges.
[Invention 1113]
said Cas12 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:27 to SEQ ID NO:37 The microfluidic cartridge of any of Inventions 1110-1112, having a sequence identity of .
[Invention 1114]
The microfluidic cartridge of any of inventions 1110-1113, wherein said Cas12 protein is selected from SEQ ID NO:27-SEQ ID NO:37.
[Invention 1115]
1111. The microfluidic cartridge of invention 1110, wherein said type V CRIPSR/Cas effector protein is Cas14 protein.
[Invention 1116]
wherein the Cas14 protein comprises a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide; A microfluidic cartridge of the present invention 1115 comprising:
[Invention 1117]
said Cas14 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:38-SEQ ID NO:129 The microfluidic cartridge of any of Inventions 1115-1116 having a sequence identity of .
[Invention 1118]
The microfluidic cartridge of any of Inventions 1115-1117, wherein said Cas14 protein is selected from SEQ ID NO:38-SEQ ID NO:129.
[Invention 1119]
1111. The microfluidic cartridge of invention 1110, wherein said type V CRIPSR/Cas effector protein is CasΦ protein.
[Invention 1120]
said CasФ protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:274 to SEQ ID NO:321 1119. A microfluidic cartridge of the invention 1119 having a sequence identity of
[Invention 1121]
The microfluidic cartridge of any of inventions 1119-1120, wherein said CasΦ protein is selected from SEQ ID NO:274-SEQ ID NO:321.
[Invention 1122]
1122. The microfluidic cartridge of any of inventions 1001-1121, further providing one or more chambers for in vitro transcription of amplified coronavirus target nucleic acid.
[Invention 1123]
1123. The microfluidic cartridge of Invention 1122, wherein said in vitro transcription comprises contacting said amplified coronavirus target nucleic acid with a reagent for in vitro transcription.
[Invention 1124]
The microfluidic cartridge of Invention 1123, wherein said reagents for in vitro transcription comprise RNA polymerase, NTPs, and primers.
[Invention 1125]
1125. The microfluidic cartridge of any of inventions 1001-1124, wherein said programmable nuclease comprises a HEPN cleavage domain.
[Invention 1126]
1126. The microfluidic cartridge of any of inventions 1001-1125, wherein said programmable nuclease is a type VI CRISPR/Cas effector protein.
[Invention 1127]
1126. The microfluidic cartridge of invention 1126, wherein said type VI CRISPR/Cas effector protein is Casl3 protein.
[Invention 1128]
The microfluidic cartridge of Invention 1127, wherein said Cas13 protein comprises a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, or a Cas13e polypeptide.
[Invention 1129]
said Cas13 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:130 to SEQ ID NO:147 The microfluidic cartridge of any of Inventions 1127-1128 having a sequence identity of .
[Invention 1130]
The microfluidic cartridge of any of Inventions 1127-1129, wherein said Cas13 protein is selected from SEQ ID NO:130-SEQ ID NO:147.
[Invention 1131]
The microfluidic cartridge of any of inventions 1001-1130, wherein said target nucleic acid is derived from a virus.
[Invention 1132]
1131. The microfluidic cartridge of Invention 1131, wherein said virus comprises a respiratory virus.
[Invention 1133]
The microfluidic cartridge of Invention 1132, wherein said respiratory virus is an upper respiratory virus.
[Invention 1134]
1131. The microfluidic cartridge of Invention 1131, wherein said virus comprises an influenza virus.
[Invention 1135]
The microfluidic cartridge of any of inventions 1131-1133, wherein said virus comprises a coronavirus.
[Invention 1136]
The microfluidic cartridge of Invention 1135, wherein said coronavirus target nucleic acid is derived from SARS-CoV-2.
[Invention 1137]
The microfluidic cartridge of any of Inventions 1135-1136, wherein said coronavirus target nucleic acid is derived from an N gene, an E gene, or a combination thereof.
[Invention 1138]
The microfluidic cartridge of any of Inventions 1135-1137, wherein said coronavirus target nucleic acid has the sequence of any one of SEQ ID NO:333-SEQ ID NO:338.
[Invention 1139]
The microfluidic cartridge of any of Inventions 1135-1138, wherein said guide nucleic acid is guide RNA.
[Invention 1140]
wherein the guide nucleic acid is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:323-SEQ ID NO:328 The microfluidic cartridge of any of Inventions 1135-1139 having a sequence identity of .
[Invention 1141]
The microfluidic cartridge of any of Inventions 1135-1140, wherein said guide nucleic acid is selected from any one of SEQ ID NO:323-SEQ ID NO:328.
[Invention 1142]
The microfluidic cartridge of any of Inventions 1001-1141 comprising a control nucleic acid.
[Invention 1143]
1142. The microfluidic cartridge of Invention 1142, wherein said control nucleic acid is present in the detection chamber.
[Invention 1144]
The microfluidic cartridge of any of inventions 1142-1143, wherein said control nucleic acid is RNaseP.
[Invention 1145]
The microfluidic cartridge of any of Inventions 1142-1144, wherein said control nucleic acid has the sequence of SEQ ID NO:379.
[Invention 1146]
wherein the guide nucleic acid is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:330 to SEQ ID NO:332 The microfluidic cartridge of any of Inventions 1142-1144 having a sequence identity of .
[Invention 1147]
The microfluidic cartridge of any of Inventions 1142-1146, wherein said guide nucleic acid is selected from any one of SEQ ID NO:330-SEQ ID NO:332.
[Invention 1148]
The microfluidic cartridge of any of Inventions 1134-1147, wherein said influenza virus comprises influenza A virus, influenza B virus, or a combination thereof.
[Invention 1149]
The microfluidic cartridge of any of inventions 1001-1148, wherein said guide nucleic acid targets multiple target sequences.
[Invention 1150]
The microfluidic cartridge of any of inventions 1001-1149, comprising a plurality of guide sequences tiled against viruses.
[Invention 1151]
The microfluidic cartridge of Invention 1150, wherein said plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen.
[Invention 1152]
1152. The microfluidic cartridge of any of inventions 1001-1151, wherein said labeled detector nucleic acid comprises a single-stranded reporter comprising a detection moiety.
[Invention 1153]
The microfluidic cartridge of Invention 1152, wherein said detection moiety is a fluorophore, a FRET pair, a fluorophore/quencher pair, or an electrochemical reporter molecule.
[Invention 1154]
The microfluidic cartridge of invention 1153, wherein said electrochemical reporter molecule comprises a species shown in FIG.
[Invention 1155]
1154. The microfluidic cartridge of any of inventions 1001-1154, wherein cleavage of said detector nucleic acid causes the labeled detector to produce a detectable signal.
[Invention 1156]
The microfluidic cartridge of invention 1155, wherein said detectable signal is a colorimetric, fluorescent, amperometric, or potentiometric signal.
[Invention 1157]
a) providing a sample from a subject;
b) adding said sample to the microfluidic cartridge of any of Inventions 1001-1156;
c) correlating the detectable signal of any of the inventions 1084-1156 with the presence or absence of said target nucleic acid; and
d) optionally quantifying said detectable signal, thereby quantifying the amount of said target nucleic acid present in said sample
A method of detecting a target nucleic acid, comprising:
[Invention 1158]
Use of the microfluidic cartridge of any of inventions 1001-1156 in a method of detecting a target nucleic acid.
[Invention 1159]
Use of the system of any of inventions 1001-1156 in a method of detecting a targeting nucleic acid.
[Invention 1160]
Use of a programmable nuclease in the method of detecting a target nucleic acid of any of the inventions 1030-1063, 1066, 1150, 1153.
[Invention 1161]
Use of any of the compositions of the invention 1066-1087 in a method of detecting a target nucleic acid.
[Invention 1162]
Use of a DNA-activated programmable RNA nuclease in a method of assaying target deoxyribonucleic acid from a virus in a sample of any of the inventions 1088, 1090-1106, or 1151.
[Invention 1163]
Use of a DNA-activated programmable RNA nuclease in a method of assaying a target ribonucleic acid from a virus in a sample of any of the inventions 1088, 1090-1106, or 1152.
[Invention 1164]
Use of a programmable nuclease in the method of detecting a target nucleic acid in a sample of any of the inventions 1108-1120, 1123-1148, or 1156.
[Invention 1165]
at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity with any one of SEQ ID NO:348 through SEQ ID NO:353 A composition comprising a non-naturally occurring nucleic acid comprising a sequence having
[Invention 1166]
at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity with any one of SEQ ID NO:354 through SEQ ID NO:359 A composition comprising a non-naturally occurring nucleic acid comprising a sequence having
[Invention 1167]
The composition of invention 1165, wherein said nucleic acid comprises a sequence selected from any one of SEQ ID NO:348-SEQ ID NO:353.
[Invention 1168]
The composition of invention 1166, wherein said nucleic acid comprises a sequence selected from any one of SEQ ID NO:354-SEQ ID NO:359.
[Invention 1169]
A composition of the invention 1165 comprising the nucleic acids of SEQ ID NO:348 to SEQ ID NO:353 and configured to be added to a single reaction chamber.
[Invention 1170]
The composition of invention 1169, wherein said single reaction chamber is an amplification chamber of any of inventions 1001-1164.
[Invention 1171]
A composition of invention 1166 comprising the nucleic acids of SEQ ID NO:354 to SEQ ID NO:359 and configured to be added to a single reaction chamber.
[Invention 1172]
The composition of invention 1171, wherein said single reaction chamber is an amplification chamber of any of inventions 1001-1164.
[Invention 1173]
The composition of any of Inventions 1165-1172 further comprising any one of the detector nucleic acids listed in Table 5.
[Invention 1174]
The composition of any of inventions 1165-1173, further comprising a coronavirus target nucleic acid.
[Invention 1175]
The composition of invention 1174, wherein said coronavirus target nucleic acid is derived from an E gene, an N gene, or a combination thereof.
[Invention 1176]
Any one of invention 1174-1175, wherein said coronavirus target nucleic acid comprises any one of SEQ ID NO:333-SEQ ID NO:338, SEQ ID NO:375-SEQ ID NO:376, or fragments thereof composition.
[Invention 1177]
The composition of any of inventions 1165-1176, further comprising a guide nucleic acid.
[Invention 1178]
wherein the guide nucleic acid is at least 80%, at least 85%, at least 90%, at least 92% with any one of SEQ ID NO:323 to SEQ ID NO:332 or SEQ ID NO:18 to SEQ ID NO:26; A composition of the invention 1084 comprising sequences having at least 95%, at least 97%, or at least 99% sequence identity.
[Invention 1179]
The composition of any of Inventions 1177-1178, wherein said guide nucleic acid is selected from SEQ ID NO:271-SEQ ID NO:273, SEQ ID NO:374, or SEQ ID NO:249-258 thing.
[Invention 1180]
The composition of any of Inventions 1165-1179, further comprising reagents for amplification.
[Invention 1181]
The composition of invention 1180, wherein said reagents for amplification comprise a polymerase and dNTPs.
[Invention 1182]
The composition of any of Inventions 1165-1181, further comprising reagents for reverse transcription.
[Invention 1183]
The composition of invention 1182, wherein said reagents for reverse transcription comprise reverse transcriptase and dNTPs.
[Invention 1184]
The composition of any of Inventions 1165-1183 further comprising a control nucleic acid.
[Invention 1185]
The composition of Invention 1184, wherein said control nucleic acid is RNase P.
[Invention 1186]
The composition of any of Inventions 1184-1185, wherein said control nucleic acid has the sequence of SEQ ID NO:379.
[Invention 1187]
The composition of any of inventions 1165-1186, further comprising a programmable nuclease.
[Invention 1188]
1187. The composition of invention 1187, wherein said programmable nuclease is a type V CRISPR/Cas effector protein.
[Invention 1189]
1188. The composition of invention 1188, wherein said type V CRISPR/Cas effector protein is Casl2 protein.
[Invention 1190]
The present invention, wherein said Cas12 protein comprises a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide. 1189 compositions.
[Invention 1191]
The composition of invention 1190, wherein said Cas12 protein is a Cas12a protein.
[Invention 1192]
said Cas12 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:27 to SEQ ID NO:37 The composition of any of Inventions 1189-1191 having the sequence identity of
[Invention 1193]
1189 of the invention, wherein said Cas12 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:37 The composition of any of -1192.
[Invention 1194]
The composition of any of inventions 1189-1193, wherein said Cas12 protein is selected from any one of SEQ ID NO:27-SEQ ID NO:37.
[Invention 1195]
The composition of any of inventions 1189-1194, wherein said Cas12 protein has the sequence of SEQ ID NO:37.
[Invention 1196]
1188. The composition of invention 1188, wherein said type V CRISPR/Cas effector protein is Casl4 protein.
[Invention 1197]
wherein the Cas14 protein comprises a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide; A composition of the present invention 1197, comprising:
[Invention 1198]
said Cas14 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:38-SEQ ID NO:129 The composition of any of the inventions 1196-1197, which has the sequence identity of
[Invention 1199]
The composition of any of inventions 1107-1109, wherein said Cas14 protein is selected from SEQ ID NO:38-SEQ ID NO:129.
[Invention 1200]
1188. The composition of any of invention 1188, wherein said type V CRISPR/Cas effector protein is CasΦ protein.
[Invention 1201]
said CasФ protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:274 to SEQ ID NO:321 The composition of the invention 1200, which has the sequence identity of
[Invention 1202]
The composition of any of inventions 1200-1201, wherein said CasΦ protein is selected from SEQ ID NO:274-SEQ ID NO:321.
[Invention 1203]
The composition of invention 1187, wherein said programmable nuclease is Cas13 protein.
[Invention 1204]
The composition of invention 1203, wherein said Cas13 protein comprises a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, or a Cas13e polypeptide.
[Invention 1205]
said Cas13 protein is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of SEQ ID NO:130 to SEQ ID NO:147 The composition of any of the invention 1203-1204, which has the sequence identity of
[Invention 1206]
The composition of any of invention 1203-1205, wherein said Cas13 protein is selected from SEQ ID NO:130-SEQ ID NO:147.
[Invention 1207]
The composition of any of Inventions 1165-1206, further comprising reagents for in vitro transcription.
[Invention 1208]
The composition of invention 1207, wherein said reagents for in vitro transcription comprise RNA polymerase and NTP.
[Invention 1209]
The composition of any of Inventions 1165-1208, further comprising a lysis buffer.
[Invention 1210]
The composition of any of Inventions 1165-1209, further comprising a reporter molecule.
[Invention 1211]
wherein the reporter molecule is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% with any one of the sequences listed in Table 12 or Table 22 A composition of the invention 1210 comprising sequences with sequence identity.
[Invention 1212]
The composition of any of Inventions 1165-1211 present in a test tube, well plate, lateral flow strip, or microfluidic cartridge.
[Invention 1213]
The composition of any of Inventions 1165-1212 present in a single volume.
[Invention 1214]
The composition of any of Inventions 1165-1213 present in separate volumes.

の成分である。図20Aは、DNA/RNA、LAMP/RT-LAMPの提供、およびCas12a検出を含むワークフローの概略図を示す。図20Bは、蛍光によって定量化される、異なる温度でのRNase P POP7のためのLAMP/RT-LAMP反応の結果に至る時間を示す。図20Cは、68°Cの温度反応からの、LAMP/RT-LAMPアンプリコンの37°CでのCas12a特異的検出を示す3つのグラフを示す。
（図２１）インフルエンザAウイルス用の、3つの異なるRT-LAMPアンプリコンの特異的検出を示す。左側は、DNA/RNA、LAMP/RT-LAMPの提供、およびCas12a検出を含むワークフローの概略図である。右側は、蛍光によって経時的に測定されたCas12a検出の、各試験条件についての結果を示すグラフである。
（図２２）インフルエンザB（IBV）RT-LAMPアンプリコンの特異的検出のための最適なcrRNAの同定を示す。左側は、DNA/RNA、LAMP/RT-LAMPの提供、およびCas12a検出を含むワークフローの概略図である。右側は、各試験条件について経時的に蛍光によって測定したCas12a検出の結果を示すグラフである（IAVはインフルエンザAウイルスであり、IBVはインフルエンザBウイルスであり、NTCはテンプレートの無い対照である）。
（図２３）RT-LAMP反応の生成物を示すバンドを有する1％アガロースゲルの結果を示す。
（図２４）インフルエンザAおよびインフルエンザBについての多重化RT-LAMP反応からのアンプリコン間のCas12a識別を示す。図24Aは、ウイルスRNA、多重化RT-LAMPの提供、およびCas12aインフルエンザA検出またはCas12aインフルエンザB検出を含むワークフローの概略図を示す。図24Bは、60°Cで30分間の多重化RT-LAMP増幅後のRT-LAMPアンプリコンのCas12a検出を示す。図24Cは、IAVおよびIBVの10,000ウイルスゲノムコピーについてRT-LAMPアンプリコンを37°Cで30分間Cas12a検出した、バックグラウンドを差し引いた蛍光を示す。
（図２５）60°Cで30分間の多重化RT-LAMP増幅後、インフルエンザA、インフルエンザB、およびマムーサス・プリミゲニウス（ケナガマンモス）ミトコンドリア内部増幅対照配列についての三つ組み多重化RT-LAMP反応間のCas12a識別を示す。上部は、ウイルスRNA、多重化RT-LAMPの提供、およびCas12aインフルエンザA検出またはCas12aインフルエンザB検出またはCas12内部増幅対照検出を含むワークフローの概略図である。下部は、蛍光によって経時的に測定されたCas12検出の、各試験条件についての結果を示すグラフである。
（図２６）LAMPおよびRT-LAMPプライマー設計の概略図を示す。図26Aは、LAMPおよびRT-LAMPで使用されるプライマーの同一性を示す概略図を示す。プライマーLFおよびLBは、いくつかのLAMPおよびRT-LAMP設計では任意選択であるが、一般に反応の効率を上げる。図26Bは、様々なLAMPプライマーにおけるT7プロモーターの位置および配向を示す概略図を示す。
（図２７）T7プロモーターが、F3もしくはB3プライマー（アウタープライマー）、またはインフルエンザAのためのFIPもしくはBIPプライマーに含まれ得ることを示す。図27Aは、DNA/RNA、LAMP/RT-LAMPの提供、インビトロ転写、およびCas13a検出を含むワークフローの概略図を示す。図27Bは、蛍光によって定量化された、異なるプライマーセットを使用したインフルエンザAのRT-LAMP反応の結果までの時間を示す。図27Cは、異なるプライマーセットを37°Cで10分間使用した、インフルエンザAについてのRT-LAMP反応の生成物のT7 RNAポリメラーゼによるインビトロ転写（IVT）を示す。
（図２８）Cas12による、インフルエンザAについてのRT-SIBAアンプリコンの検出を示す。左側は、DNA/RNA、SIBA/RT-SIBAの提供、およびCas12a検出を含むワークフローの概略図である。右側は、試験した条件のそれぞれについて蛍光によって測定したCas12a検出を示すグラフである。
（図２９）典型的なレポーターを含む、Mileniaの市販ストリップのレイアウトを示す。
（図３０）アンプリコンを含む、Milenia HybridDetect 1ストリップのレイアウトを示す。
（図３１）標準的なCasレポーターを含む、Milenia HybridDetect 1ストリップのレイアウトを示す。
（図３２）FAM分子（緑色のスタートで示す）に結合したビオチン-dT（ピンクの六角形で示す）へのDNAリンカーを含む改変Casレポーターを示す。
（図３３）改変Casレポーターを含む、Milenia HybridDetectストリップのレイアウトを示す。
（図３４）単一の標的アッセイフォーマット（左）および多重化アッセイフォーマット（右）の一例を示す。
（図３５）使用前のアッセイ（上）、陽性結果を有するアッセイ（左中央）、陰性結果を有するアッセイ（右中央）、および失敗した試験（下）の別のバリエーションを示す。
（図３６）つながれたラテラルフローCasレポーターの1つの設計を示す。
（図３７）磁気ビーズを使用するつながれた切断レポーターを使用する、CRISPR診断のためのワークフローを示す。
（図３８）反応チャンバに到達する前にストレプトアビジン-ビオチンによって濾過される酵素-レポーター系の概略図を示す。
（図３９）標的核酸の検出に使用されるインベルターゼ核酸を示す。インベルターゼ-核酸は、磁気ビーズ上に固定化されており、Casタンパク質、ガイドRNAおよび標的核酸を含む試料反応に添加される。標的認識は、Casタンパク質を活性化して、インベルターゼ-核酸の核酸を切断し、固定化された磁気ビーズからインベルターゼ酵素を遊離させる。この溶液は、スクロースおよびDNS試薬を含有し、インベルターゼがスクロースをグルコースに変換すると黄色から赤色に変色する「反応混合物」に移されるか、またはデジタル読み出しのために手持ち式血糖値測定器装置に移すことができる。
（図４０）ツーポットDETECTRアッセイの1つのレイアウトを示す。このレイアウトでは、スワブ収集キャップがスワブ貯蔵器チャンバを密閉する。スワブ貯蔵器チャンバに対して時計回りに、増幅反応混合物を保持するチャンバがある。増幅反応混合物を保持するチャンバに対して時計回りに、DETECTR反応混合物を保持するチャンバがある。これに対して時計回りに検出領域がある。検出領域に対して時計回りに、pHバランスウェルがある。カートリッジウェルのキャップが示され、様々な試薬混合物を含有するすべてのウェルを封止している。カートリッジ自体は、概略図の下部に正方形の層として示されている。右側には、各チャンバ/ウェル内の流体を駆動し、カートリッジ全体に接続される、機器パイパーポンプの図が示されている。カートリッジの下方には、機器と接続する回転バルブがある。
（図４１）図40のツーポットDETECTRアッセイにおける様々な反応の一ワークフローを示す。最初に、左上の図に示すように、スワブが200ulスワブチャンバに挿入され、混合され得る。中央の左の図では、バルブを時計回りに「スワブチャンバ位置」まで回転させ、1uLの試料を採取する。左下の図では、バルブを時計回りに「増幅反応混合」位置まで回転させ、1ulの試料を分注して混合する。右上の図では、2uLの試料が「増幅反応混合」から吸引される。上の中央の図では、バルブを時計回りに「DETECTR」位置まで回転させ、試料を分注して混合し、20ulの試料が吸引される。最後に、右下の図では、バルブを時計回りに検出領域位置まで回転させ、20ulの試料を分注する。
（図４２）本開示の方法およびシステムとも一致する、図41に示されるワークフローの変形例を示す。左側は、図41の右上に示されている図である。右側は修正図であり、そこでは、スワブ溶解チャンバに対して反時計回りに第1の増幅チャンバがあり、スワブ溶解チャンバに対して時計回りに第2の増幅チャンバがある。さらに、増幅チャンバ＃2に対して時計回りに2つのセット、すなわちそれぞれ「二重DETECTRチャンバ＃2」および「二重DETECTRチャンバ＃1」と標識された「二重」DETECTRチャンバがある。
（図４３）図42に示す変更されたレイアウトのワークフローの内訳を示す。具体的には、200ulの試料を保持するスワブ溶解チャンバから、20ulの試料を増幅チャンバ＃1に移動させることができ、20ulの試料を増幅チャンバ＃2に移動させることができる。増幅チャンバ＃1で増幅した後、20ulの試料を二重DETECTRチャンバ＃1aに移動させ、20ulの試料を二重DETECTRチャンバ＃1bに移動させることができる。さらに、増幅チャンバ＃2で増幅した後、20ulの試料を二重DETECTRチャンバ＃2aに移動させ、20ulの試料を二重DETECTRチャンバ＃2bに移動させることができる。
（図４４）図43および図42に示すカートリッジの変形例を示す。
（図４５）図44のカートリッジの上面図を示す。このレイアウトおよびワークフローは、図40～図41のレイアウトおよびワークフローと比較する再現を有する。
（図４６）ツーポットDETECTRアッセイのレイアウトを示す。上部には、カートリッジと接続する空気圧ポンプが示されている。中央には、貯蔵器を有する最上層を示すカートリッジの上面図が示されている。下部には、試料を含むスライドバルブ、および左側の溶解チャンバを指す矢印が示され、右側に増幅チャンバ、さらに右側にDETECTチャンバが続く。
（図４７）本明細書に開示されるDETECTRアッセイと、標的核酸を検出するゴールドスタンダードPCRベース方法との比較を示す。粗（左）から純粋（右）への試料調製評価の勾配を示すフローチャートが示されている。粗試料を純粋な試料にする試料調製工程には、溶解、結合、洗浄および溶出が含まれる。本明細書に開示されるDETECTRアッセイは、溶解の試料調製工程のみを必要とし、粗試料をもたらし得る。一方、PCRベース方法は、溶解、結合、洗浄および溶出を必要とし、非常に純粋な試料をもたらし得る。
（図４８）標的RT-LAMP DNAアンプリコンのCas13a検出を示す。図48Aは、DNA/RNA、LAMP/RT-LAMPの提供、およびCas13a検出を含むワークフローの概略図を示す。図48Bは、y軸上のバックグラウンドを差し引いた蛍光によって測定される、第1のプライマーセットを含む標的RT-LAMP DNAアンプリコンのCas13a特異的検出を示す。図48Cは、y軸上のバックグラウンドを差し引いた蛍光によって測定される、第2のプライマーセットを含む標的RT-LAMP DNAアンプリコンのCas13a特異的検出を示す。
（図４９）図49Aは、2.5nMのRNA、一本鎖DNA（ssDNA）または二本鎖（dsDNA）を標的核酸として使用するCas13検出アッセイを示し、検出は試験した標的核酸のそれぞれについて蛍光によって測定した。図48Bは、2.5nMのRNA、ssDNAおよびdsDNAを標的核酸として使用するCas12検出アッセイを示し、検出は試験した標的核酸のそれぞれについて蛍光によって測定した。図48Cは、様々な濃度の標的RNA、標的ssDNAおよび標的dsDNAに対するCas13およびCas12の性能を示し、試験した標的核酸のそれぞれについて検出を蛍光によって測定した。
（図５０）170nMの様々なレポーター基材を含む2.5nMの標的ssDNAを使用するLbuCas13a検出アッセイを示し、試験したレポーター基材のそれぞれについて蛍光によって検出を測定した。
（図５１）図51Aは、10nMまたは0nMの標的RNAを使用するLbuCas13a（SEQ ID NO:131）およびLwaCas13a（SEQ ID NO:137）についてのCas13検出アッセイの結果を示す。検出は、レポーターの切断から生じる蛍光によって経時的に測定した。図51Bは、10nMまたは0nMの標的ssDNAを使用するLbuCas13a（SEQ ID NO:131）およびLwaCas13a（SEQ ID NO:137）についてのCas13検出アッセイの結果を示す。検出は、レポーターの切断から生じる蛍光によって経時的に測定した。
（図５２）6.8～8.2の範囲の様々なpH値を有する緩衝液中で1nMの標的RNA（左）または標的ssDNA（右）を使用するLbuCas13a（SEQ ID NO:131）検出アッセイを示す。
（図５３）図53Aは、1ヌクレオチド間隔で標的配列に沿ってタイリングされたガイドRNA（gRNA）を示す。図53Bは、1ヌクレオチド間隔でタイリングされたgRNAおよび標的外gRNAを含む0.1nM RNAまたは2nM標的ssDNAを使用した、LbuCas13a（SEQ ID NO:131）検出アッセイを示す。図53Cは、標的ssDNAの性能によってランク付けされた図97Bからのデータを示す。図53Dは、標的RNAの3’末端上の各ヌクレオチドに対するgRNAの性能を示す。図53Eは、標的ssDNAの3’末端上の各ヌクレオチドに対するgRNAの性能を示す。
（図５４）図54Aは、様々なLAMP等温核酸増幅反応からの1μLの標的DNAアンプリコンを使用したLbuCas13a検出アッセイを示す。図54Bは、様々な量のPCR反応を標的DNAとして使用するLbuCas13a（SEQ ID NO:131）検出アッセイを示す。
（図５５）DETECTRアッセイのための空気圧バルブ装置レイアウトを示す。図55Aは、空気圧バルブ装置の概略図を示す。ピペットポンプは、試料を吸引して分注する。空気マニホールドを空気圧ポンプに接続して、常閉バルブを開閉する。空気圧装置は、流体をある位置から次の位置に移動させる。空気圧の設計は、他の装置の設計と比較してチャネルクロストークが低減されている。図55Bは、図55Aに示す振動バルブ空気圧装置で使用するためのカートリッジの概略図を示す。バルブ構成が示されている。常閉バルブ（そのようなバルブの1つを矢印で示す）は、チャンバが使用されていないときに各チャンバをシステムの残りの部分から隔離するために、チャネルの上部にエラストマー封止を備える。空気圧ポンプは空気を使用して、カートリッジ内の必要なチャンバに流体を移動させるために必要に応じてバルブを開閉する。
（図５６）図55Aに示す空気圧バルブ装置のバルブ回路レイアウトを示す。（i）に示すように、すべてのバルブを閉じた状態で、試料を試料ウェルに入れる。試料を試料ウェルに溶解する。溶解された試料は、（ii）に示すように、第1の振動バルブを開くことによって試料チャンバから第2のチャンバに移され、ピペットポンプを用いて試料を吸引する。次いで、試料は、（iii）に示すように、第1の振動バルブを閉じ、第2の振動バルブを開くことによって第1の増幅チャンバに移され、そこで増幅混合物と混合される。試料は増幅混合物と混合した後、（iv）に示すように、第2の振動バルブを閉じ、第3の振動バルブを開くことによって、次のチャンバに移動させる。試料は、（v）に示されるように、第3の振動バルブを閉じ、第4の振動バルブを開くことによってDETECTRチャンバに移動される。試料は、（vi）に示すように、常開（例えば、振動タイプ）バルブの異なる系列を開閉することによって、チャンバの異なる系列を通って移動させることができる。所望のチャンバ系列内の個々のバルブの作動は、チャネル間の交差汚染を防止する。
（図５７）スライドバルブ装置の概略図を示す。チャネルのオフセットピッチは、吸引および各ウェルへの別々の分注を可能にし、増幅チャンバと対応するチャンバとの間のクロストークを緩和するのに役立つ。
（図５８）図57に示すスライドバルブ装置を通る試料移動の図を示す。最初の閉位置（i）において、試料を試料ウェルに充填し、溶解する。次いで、スライドバルブを器具によって作動させ、適切な量をチャネルに分注するピペットポンプを使用して試料を各チャネルに充填する（ii）。試料は、スライドバルブを作動させることによって増幅チャンバに送達され、ピペットポンプで混合される（iii）。増幅チャンバからの試料は、各チャネルに吸引され（iv）、次いで、スライドバルブおよびピペットポンプを作動させることによって、各DETECTRチャンバに分注され、混合される（v）。
（図５９）本開示の空気圧バルブ装置のカートリッジの最上層の概略図を、適切な寸法を強調して示す。概略図は、2インチ×1.5インチの1つのカートリッジを示す。
（図６０）電気化学的寸法に適応させた、本開示の空気圧バルブ装置のカートリッジの変更された最上層の概略図を示す。この概略図では、3つの線が検出チャンバ（最も右側の4つのチャンバ）に示されている。これらの3本の線は、3電極システムを形成するために使い捨てカートリッジ内に共成形、3D印刷、または手動で組み立てられる配線（または「金属リード」）を表す。
（図６１）標的核酸配列のループ介在等温増幅（LAMP）用のプライマーを設計するためのスキームを示す。「c」を表示する領域は、「c」を表示しない対応する領域と逆相補的である（例えば、領域F3cは領域F3と逆相補的である）。
（図６２）LAMPプライマーに対応するか、もしくはアニールする核酸配列、またはガイドRNA配列、またはプロトスペーサー隣接モチーフ（PAM）もしくはプロトスペーサー隣接部位（PFS）を含むもの、ならびにLAMPおよびDETECTRによる増幅および検出のための標的核酸配列の様々な領域の例示的な構成の概略図を示す。図62Aは、LAMPプライマーに対応するか、またはアニールする核酸配列の様々な領域に対するガイドRNA（gRNA）の例示的な配置の概略図を示す。この配置では、ガイドRNAは、F1c領域（すなわち、F1領域と逆相補的な領域）とB1領域との間に存在する標的核酸の配列に逆相補的である。図62Bは、LAMPプライマーに対応するか、またはアニールする核酸配列の様々な領域に対するガイドRNA配列の例示的な配置の概略図を示す。この配置では、ガイドRNAは、F1c領域とB1領域との間に存在する標的核酸の配列に部分的に逆相補的である。例えば、標的核酸は、ガイド核酸の少なくとも60％に逆相補的である、F1c領域とB1領域との間の配列を含む。この配置では、ガイドRNAは、図40に示すフォワードインナープライマーまたはバックワードインナープライマーと逆相補的ではない。図62Cは、LAMPプライマーに対応するか、またはアニールする核酸配列の様々な領域に対するガイドRNAの例示的な配置の概略図を示す。この配置では、ガイドRNAは、B1領域とB2領域との間のループ領域内に存在する標的核酸の配列にハイブリタイズする。フォワードインナープライマー、バックワードインナープライマー、フォワードアウタープライマー、およびバックワードアウタープライマー配列は、PAMまたはPFSを含有せず、PAMまたはPFSと逆相補的ではない。図62Dは、LAMPプライマーに対応するか、またはアニールする核酸配列の様々な領域に対するガイドRNAの例示的な配置の概略図を示す。この配置では、ガイドRNAは、F2c領域とF1c領域との間のループ領域内に存在する標的核酸の配列にハイブリタイズする。プライマー配列は、PAMもPFSも含まず、PAMともPFSとも逆相補的ではない。
（図６３）LAMPプライマーもしくはガイドRNA配列に対応するか、もしくはアニールする、またはプロトスペーサー隣接モチーフ（PAM）もしくはプロトスペーサー隣接部位（PFS）を含む核酸配列、ならびに増幅および検出のために組み合わせたLAMPおよびDETECTRについての標的核酸配列の様々な領域の例示的な構成の概略図をそれぞれ示す。右側では、概略図はまた、LAMP増幅およびガイドRNA配列を使用して標的核酸配列の存在を検出する、対応する蛍光データを示し、ここで蛍光信号は、DETECTR反応の出力であり、標的核酸の存在を示す。図63Aは、LAMPプライマーに対応またはアニールする核酸配列の様々な領域の配置、およびLAMPプライマーに対する3つのガイドRNA（gRNA1、gRNA2およびgRNA3）の位置の概略図を示す（左）。gRNA1はB2c領域と重複し、したがってB2領域と逆相補的である。gRNA2はB1領域と重複し、したがってB1c領域と逆相補的である。gRNA3はB3領域と部分的に重複し、かつB2領域と部分的に重複し、したがって、B3c領域と部分的に逆相補的であり、かつB2c領域と部分的に逆相補的である。相補的領域（B1、B2c、B3c、F1、F2c、およびF3c）は図示されていないが、図40に示されている領域に対応する。右側は、標的核酸の10,000ゲノムコピーまたは標的核酸の0ゲノムコピーの存在下でのDETECTR反応からの蛍光のグラフである。図63Bは、LAMPプライマーに対応またはアニールする核酸配列の様々な領域の配置、およびLAMPプライマーに対する3つのガイドRNA（gRNA1、gRNA2およびgRNA3）の位置の概略図を示す（左）。gRNA1はB1c領域と重複し、したがってB1領域と逆相補的である。gRNA2はLF領域と重複し、したがってLFc領域と逆相補的である。gRNA3はB2領域と部分的に重複し、かつLBc領域と部分的に重複し、したがって、B2c領域と部分的に逆相補的であり、かつLB領域と部分的に逆相補的である。右側は、標的核酸の10,000ゲノムコピーまたは標的核酸の0ゲノムコピーの存在下でのDETECTR反応からの蛍光のグラフである。図63Cは、LAMPプライマーに対応またはアニールする核酸配列の様々な領域の配置、およびLAMPプライマーに対する3つのガイドRNA（gRNA1、gRNA2およびgRNA3）の位置の概略図を示す（左）。gRNA1はB1c領域と重複し、したがってB1領域と逆相補的である。gRNA2はLF領域と部分的に重複し、かつF2c領域と部分的に重複し、したがってLFc領域と部分的に逆相補的であり、かつF2領域と部分的に逆相補てきである。gRNA3はB2領域と重複し、したがってB2c領域と逆相補的である。右側は、標的核酸の10,000ゲノムコピーまたは標的核酸の0ゲノムコピーの存在下でのDETECTR反応からの蛍光のグラフである。
（図６４Ａ）LAMPプライマーもしくはガイドRNA配列に対応もしくはアニールする、またはプロトスペーサー隣接モチーフ（PAM）もしくはプロトスペーサー隣接部位（PFS）を含む核酸配列、ならびに図63Aに示すLAMPおよびDETECTRアッセイについての標的核酸配列の様々な領域の配置および配列の詳細な内訳を示す。図64AはSEQ ID NO：393を開示している。
（図６４Ｂ）LAMPプライマーもしくはガイドRNA配列に対応もしくはアニールする、またはプロトスペーサー隣接モチーフ（PAM）もしくはプロトスペーサー隣接部位（PFS）を含む核酸配列、ならびに図63Bに示すLAMPおよびDETECTRアッセイについての標的核酸配列の様々な領域の配置および配列の詳細な内訳を示す。図64BはSEQ ID NO：393を開示している。

（図６４Ｃ）LAMPプライマーもしくはガイドRNA配列に対応もしくはアニールする、またはプロトスペーサー隣接モチーフ（PAM）もしくはプロトスペーサー隣接部位（PFS）を含む核酸配列、ならびに図63Cに示すLAMPおよびDETECTRアッセイについての標的核酸配列の様々な領域の配置および配列の詳細な内訳を示す。図64CはSEQ ID NO：393を開示している。
（図６５）DNA結合色素を使用して検出された逆転写LAMP（RT-LAMP）反応の結果に至る時間を示す図である。
（図６６）RT-LAMP反応からの生成物との5分間のインキュベーション後のDETECTR反応からの蛍光信号を示す。図65のLAMPプライマーセット＃1～6は、ガイドRNA＃2（SEQ ID NO:250）と共に使用するように設計され、LAMPプライマーセット＃7～10は、ガイドRNA＃1（SEQ ID NO:249）と共に使用するように設計された。
（図６７）LAMPプライマーセット1、2、4、5、6、7、8、9、10、または陰性対照を用いたRT-LAMP増幅後の、SYTO 9（DNA結合色素）を使用したインフルエンザAウイルス（IAV）由来の配列の検出を示す。
（図６８）RT-LAMP増幅後のインフルエンザBウイルス（IBV）標的配列の増幅までの時間を示す。増加する濃度の標的配列（反応あたり標的配列の0、100、1000、10,000または100,000ゲノムコピー）の存在下でSYTO 9を使用して増幅を検出した。
（図６９）異なるプライマーセットを用いたLAMP増幅後のIAV標的配列の増幅までの時間を示す。
（図７０）LAMPプライマーセット1、2、4、5、6、7、8、9、10、または陰性対照を用いたRT-LAMP増幅後の、DETECTRを使用したインフルエンザAウイルス（IAV）由来の標的核酸配列の検出を示す。プライマーセットごとに10回の反応を行った。DETECTR信号を、反応中に存在する標的配列の量の関数として測定した。
（図７１）標的核酸配列のLAMP増幅および標的核酸配列内の一塩基多型（SNP）の検出のためのプライマーを設計するためのスキームを示す。例示的な配置では、標的核酸のSNPは、F1c領域とB1領域との間に位置する。
（図７２）標的核酸のLAMP増幅およびDETECTRを使用した標的核酸の検出の方法のための、LAMPプライマー、ガイドRNA配列、プロトスペーサー隣接モチーフ（PAM）またはプロトスペーサー隣接部位（PFS）、およびSNPを含む標的核酸の例示的な配置の概略図を示す。図72Aは、LAMPプライマーに対応するかまたはアニールする核酸配列の様々な領域に対する、ガイドRNAの例示的な配置の概略図を示す。この配置では、標的核酸のPAMまたはPFSは、F1c領域とB1領域との間に位置する。ガイドRNA配列の全体は、F1c領域とB1c領域との間に存在し得る。SNPは、ガイドRNAにハイブリダイズする標的核酸の配列内に位置するものとして示されている。図72Bは、LAMPプライマーに対応するかまたはアニールする核酸配列の様々な領域に対する、ガイドRNA配列の例示的な配置の概略図を示す。この配置では、標的核酸のPAMまたはPFSは、F1c領域とB1領域との間に配置され、標的核酸は、ガイド核酸の少なくとも60％に逆相補的なF1c領域とB1領域との間の配列を含む。この例では、ガイドRNAは、フォワードインナープライマーまたはバックワードインナープライマーと逆相補的ではない。SNPは、ガイドRNAにハイブリダイズする標的核酸の配列内に位置するものとして示されている。図72Cは、LAMPプライマーに対応するかまたはアニールする核酸配列の様々な領域に対する、ガイドRNA配列の例示的な配置の概略図を示す。この配置では、標的核酸のPAMまたはPFSは、F1c領域とB1領域との間に位置し、ガイドRNA配列全体は、F1c領域とB1領域との間に存在してもよい。SNPは、ガイドRNAにハイブリダイズする標的核酸の配列内に位置するものとして示されている。
（図７３）2つのPAM部位およびHERC2 SNPを含む核酸の例示的な配列（SEQ ID NO:394）を示す。
（図７４）LAMP増幅後の第1のPAM部位に対する9位、または第2のPAM部位に対する14位のHERC2 SNPを検出するためのDETECTR反応の結果を示す。標的配列の検出を示す蛍光信号を、HERC2中のG対立遺伝子またはA対立遺伝子のいずれかを含む標的配列の存在下で経時的に測定した。ガイドRNA（crRNAのみ）を使用して標的配列を検出して、A対立遺伝子またはG対立遺伝子のいずれかを検出した。
（図７５）標的核酸配列のLAMP増幅後のDETECTR反応からの蛍光のヒートマップを示す。DETECTR反応は、A対立遺伝子（SEQ ID NO:255、「R570 A SNP」）またはG SNP対立遺伝子（SEQ ID NO:256、「R571 G SNP」）に特異的なガイドRNA（crRNAのみ）を使用して、2つのHERC2対立遺伝子間を識別した。陽性検出は、DETECTR反応における高い蛍光値によって示される。
（図７６）LAMPによる標的核酸の複合LAMP増幅およびDETECTRによる標的核酸の検出を示す図である。検出は、赤色LEDで試料を照らすことによって、DETECTRで視覚的に行った。各反応は、青色眼表現型（「青色眼」）または褐色眼表現型（「褐色眼」）のいずれかのSNP対立遺伝子を含む標的核酸配列を含有した。試料「褐色＊」および「青色＊」は、それぞれ、A対立遺伝子陽性対照およびG対立遺伝子陽性対照であった。各LAMP DETECTR反応には、褐色眼表現型（「Br」）または青色眼表現型（「Bl」）のいずれかのためのガイドRNAを使用した。
（図７７）逆転写ループ介在等温増幅（RT-LAMP）を使用して試料中のSARS-CoV-2（「2019-nCoV」）の有無を調製および検出する工程を概略的に示し、Cas12は、SARS-CoV-2（「2019-nCoV反応」）の有無を調製および検出する工程を概略的に示す。
（図７８）異なるプライマーセット（「2019-nCoV-set1」～「2019-nCoV-set12」）で増幅し、LbCas12aおよびSARS-CoV-2のN遺伝子に指向するgRNAを用いて検出したSARS-CoV-2 N遺伝子のDETECTRアッセイ結果を示す。結果までの時間が短いほど、肯定的な結果を示す。すべてのプライマーセットについて、結果までの時間は、試料に含まれる標的配列が多いほど短くなり、アッセイが標的配列に対して感受性であったことを示している。
（図７９）0fMおよび5fMの試料について図78にプロットされたDETECTR反応の個々のトレースを示す。各プロットにおいて、0fMトレースはベースラインの上には見えず、非特異的検出がほとんどないか全くないことを示している。
（図８０）N遺伝子、E遺伝子、または標的なし（「NTC」）のいずれかを含有し、SARS-CoV-2のE遺伝子に指向するプライマーセット（「2019-nCoV-E-set13」から「2019-nCoV-E-set20」）またはSARS-CoV-2のN遺伝子する指向するプライマーセット（2019-nCoV-N-set21」から「2019-nCoV-N-set24」）を使用して増幅された試料における、DETECTR反応の結果が得られるまでの時間を示す。SARS-CoV-2 E遺伝子の特異的検出に最も性能の良いプライマーセットは、SARS-CoV-2-E-set14であった。
（図８１）プライマーセット1（「2019-nCoV-set1」）で増幅され、LbCas12a、およびSARS-CoV-2のN遺伝子に指向するgRNA（「R1763-CDC-N2-Wuhan」）またはSARS-CoVのN遺伝子に指向するgRNA（「R1766-CDC-N2-SARS」）のいずれかを使用して検出されたSARS-CoV-2 N遺伝子のDETECTRアッセイ結果を示す。
（図８２）SARS-CoV-2のN遺伝子に指向するプライマーセット（「2019-nCoV-N-set1」）を使用して増幅された、DETECTR反応におけるSARS-CoV-2の検出限界を決定するためのDETECTR反応の結果を示す。SARS-CoV-2 N遺伝子標的核酸のコピーを15,000、4,000、1,000、500、200、100、50、20、または0のいずれかで含有する試料を検出した。N遺伝子RNAのゲルを以下に示す。
（図８３）POP7試料プライマーセットを使用したRNase Pの増幅を示す。試料を、LAMPを使用して増幅した。DETECTR反応は、RNase Pに指向するgRNA（「R779」）およびCas12変異体（SEQ ID NO:37）を使用して行った。試料は、HeLaトータルRNAまたはHeLaゲノムDNAのいずれかを含有していた。
（図８４）多重化されたDETECTR反応の結果までの時間を示す。試料は、SARS-CoV-2のインビトロ転写されたN遺伝子（「N-gene IVT」）、SARS-CoV-2のインビトロ転写されたE遺伝子（「E-gene IVT」）、HeLaトータルRNA、または標的なし（「NTC」）のいずれかを含有した。試料を、SARS-CoV-2 N遺伝子（「set1」）、SARS-CoV-2 E遺伝子（「set14」）、またはRNase（「RNaseP」）に指向する1つまたは複数のプライマーセットを使用して増幅した。
（図８５）SARS-CoV-2 N遺伝子（「set1」）、SARS-CoV-2 E遺伝子（「set14」）、またはRNase P（「RNaseP」）のいずれかを指向するプライマーセットの異なる組み合わせを用いた多重化DETECTR反応の結果までの時間を示す。SARS-CoV-2のインビトロ転写されたN遺伝子（左、「N-gene IVT」）またはSARS-CoV-2のインビトロ転写されたE遺伝子（右、「E-gene IVT」）を含有する試料を試験した。
（図８６）図84および図85からの最も優れた性能のプライマーセットの組み合わせを用いた多重化DETECTR反応の結果までの時間を示す。
（図８７Ａ）SARS-CoV-2のN-2遺伝子を検出するために使用されるCDC-N2標的部位の配列を概略的に示す。図87Aは、SEQ ID NO：395～398をそれぞれ記載の順に開示している。
（図８７Ｂ）SARS-CoV-2 N遺伝子（「N-Sarbeco」）標的部位の領域の配列を概略的に示す。図87Bは、SEQ ID NO：399～400および400～401をそれぞれ記載の順に開示している。
（図８８）SARS-CoV-2のN遺伝子（「N-2019-nCoV」）、SARS-CoVのN遺伝子（「N-SARS-CoV」）またはbat-SL-CoV45のN遺伝子（「N-bat-SL-CoV45」）のいずれかを含む試料について、SARS-CoV-2のN遺伝子（「R1763」）、SARS-CoVのN遺伝子（「R1766」）またはサルベココロナウイルスのN遺伝子（「R1767」）のいずれかに指向するgRNAの感受性を決定するためのDETECTRアッセイの結果を示す。
（図８９）SARS-CoV-2 E遺伝子（「E-Sarbeco」）標的部位の領域の配列を概略的に示す。図89は、SEQ ID NO：402～403および403～404をそれぞれ記載の順に開示している。
（図９０）SARS-CoV-2のE遺伝子（「E-2019-nCoV」）、SARS-CoVのE遺伝子（「E-SARS-CoV」）、bat-SL-CoV45のE遺伝子（「E-bat-SL-CoV45」）またはbat-SL-CoV21のE遺伝子（「E-bat-SL-CoV21」）のいずれかを含む試料について、コロナウイルスN遺伝子に指向する2つのgRNAの感受性を決定するためのDETECTRアッセイの結果を示す。
（図９１）Cas12変異体（SEQ ID NO:37）を使用してSARS-CoV-2 N遺伝子標的RNAの存在または非存在を検出するためのラテラルフローDETECTR反応の結果を示す。ラテラルフロー試験ストリップを示す。インビトロ転写されたSARS-CoV-2 N遺伝子RNA（「N-gene IVT」）を含有する（「＋」）かまたは欠く（「-」）試料を試験した。横線の上部セット（「試験」と表記）は、DETECTR反応の結果を示した。
（図９２）プログラマブルヌクレアーゼを使用した標的核酸の検出を概略的に示す。簡潔には、トランス付帯的切断活性を有するCasタンパク質が、ガイド核酸およびガイド核酸の領域に逆相補的な標的配列に結合すると活性化される。活性化されたプログラマブルヌクレアーゼは、レポーター核酸を切断し、それによって、検出可能な信号を生成する。
（図９３）試料中の標的核酸の存在または非存在の検出を概略的に示す。試料中の選択核酸は、等温増幅を用いて増幅される。増幅された試料を、図17に示されるように、プログラマブルヌクレアーゼ、ガイド核酸およびレポーター核酸に接触させる。試料が標的核酸を含む場合、検出可能な信号が生成される。
（図９４）試料中のSARS-CoV-2（「2019-nCoV」）RNAの存在または非存在を検出するためのDETECTRラテラルフロー反応の結果を示す。RNase Pの検出を試料の品質管理として使用する。試料をインビトロで転写および増幅し（左）、Cas12プログラマブルヌクレアーゼを用いて検出した（右）。インビトロ転写されたSARS-CoV-2 RNA（「2019-nCoV IVT」）を含有する（「＋」）または欠く（「-」）試料を、Cas12プログラマブルヌクレアーゼ、およびSARS-CoV-2に指向するgRNAを用いて0分間または5分間アッセイした。反応は、SARS-CoV-2を含有する試料に対して感受性であった。
（図９５）LbCas12aプログラマブルヌクレアーゼ（SEQ ID NO:27）を使用して、患者試料中のSARS-CoV-2の存在または非存在を決定するDETECTR反応の結果を示す。
（図９６）患者試料中のSARS-CoV-2の存在または非存在を検出するための、ラテラルフローDETECTR反応の結果を示す。試料を、SARS-CoV-2に指向するgRNAまたはRNase Pに指向するgRNAのいずれかを用いて検出した。
（図９７）逆転写およびループ介在等温増幅（RT-LAMP）およびCas12検出を用いたコロナウイルスの検出のための技術仕様およびアッセイ条件を示す。
（図９８）LbCas12aを使用してSARS-CoV-2を検出するための複数のgRNAを評価するDETECTRアッセイの結果を示す。標的核酸配列を、SARS-CoV-2 E遺伝子（「2019-nCoV-E-set13」から「2019-nCoV-E-set20」またはSARS-CoV-2 N遺伝子（「2019-nCoV-N-set21」から「2019-nCoV-N-set24」）を増幅するためのプライマーセットを使用して増幅した。
（図９９）コロナウイルスの3つの異なる株、SARS-CoV-2（「COVID-2019」）、SARS-CoV、またはbat-SL-CoV45の識別における有用性について複数のgRNAを評価する、DETECTRアッセイの結果を示す。SARS-CoV-2（「N-2019-nCoV」）、SARS-CoV（「N-SARS-CoV」）、またはbat-SL-CoV45（「N-bat-SL-CoV45」）のいずれかのN遺伝子アンプリコンを含有する試料を試験した。
（図１００）コロナウイルスの3つの異なる株、SARS-CoV-2（「COVID-2019」）、SARS-CoV、またはbat-SL-CoV45の識別における有用性について複数のgRNAを評価する、DETECTRアッセイの結果を示す。SARS-CoV-2（「N-2019-nCoV」）、SARS-CoV（「N-SARS-CoV」）、またはbat-SL-CoV45（「N-bat-SL-CoV45」）のいずれかのE遺伝子アンプリコンを含有する試料を試験した。
（図１０１）LAMPプライマーセットをSARS-CoV-2標的の多重化増幅におけるそれらの有用性について評価する、DETECTRアッセイの結果を示す。試料を、SARS-CoV-2 N遺伝子（「set1」）またはSARS-CoV-2 E遺伝子（「set14」）、またはRNase P（「RNaseP」）に指向する1つまたは複数のプライマーセットを用いて増幅した。
（図１０２）一般的な試料緩衝液に対するRT-LAMP増幅反応の感受性を評価するDETECTRアッセイの結果を示す。様々な緩衝希釈度（左から右へ:1×、0.5×、0.25×、0.125×、または緩衝液なし）の、ユニバーサルトランスポート培地（UTM、上部）またはDNA/RNA Shield緩衝液（下）中で反応を測定した。
（図１０３）SARS-CoV-2（COVID-19感染症に起因するウイルス）についてのDETECTRアッセイの検出限界（LoD）を決定するためのDETECTRアッセイの結果を示す。
（図１０４）LbCas12aプログラマブルヌクレアーゼ（SEQ ID NO:27）を使用した2-plex多重化RT-LAMP反応において、SARS-CoV-2のN遺伝子（「R1763-N-gene」）に指向するgRNAの標的特異性を評価するDETECTRアッセイの結果を示す。
（図１０５）LbCas12aプログラマブルヌクレアーゼ（SEQ ID NO:27）を使用した3-plex多重化RT-LAMP反応において、SARS-CoV-2のN遺伝子（「R1763-N-gene」）またはSARS-CoV-2のE遺伝子（「R1765-E-gene」）に指向するgRNAの標的特異性を評価するDETECTRアッセイの結果を示す。
（図１０６）PCRDラテラルフロー装置に適合するディテクター核酸の設計を示す。例示的な適合ディテクター核酸である、rep072、rep076、およびrep100が提供される（左）。これらのディテクター核酸をPCRDラテラルフロー装置（右）で使用して、標的核酸の存在または非存在を検出することができる。右上の概略図は、標的核酸を含有しない試料と接触させた場合に生成される例示的なバンド構成を示す。右下の概略図は、標的核酸を含有する試料と接触させた場合に生成される例示的なバンド構成を示す。図106は、SEQ ID NO：9、372、および372をそれぞれ記載の順に開示している。
（図１０７）図107Aは、コロナウイルスのゲノム内のE（エンベロープ）遺伝子およびN（核タンパク質）遺伝子領域の位置を示すゲノムマップを示す。EおよびN遺伝子領域に関するプライマーおよびプローブの対応する領域またはアニーリング領域を、それぞれの遺伝子領域の下に示す。RT-LAMPプライマーは黒い長方形で示されており、FIPプライマー（灰色）のF1cとB1cの半分との結合位置は破線の境界を有する縞模様の長方形で表されている。世界保健機関（WHO）および疾病管理センター（CDC）によって用いられる試験で増幅された領域は、それぞれ「WHO Eアンプリコン」および「CDC N2アンプリコン」と呼ばれる。図107Bは、LbCas12aプログラマブルヌクレアーゼ（SEQ ID NO:27）を使用して、様々なコロナウイルス株（SARS-CoV-2、SARS-CoV、またはbat-SL-CoVZC45）のN遺伝子またはE遺伝子に指向するgRNAの特異性または広範な検出有用性を評価するDETECTRアッセイの結果を示す。アッセイで使用したN遺伝子gRNA（左、「N遺伝子」）は、SARS-CoV-2に特異的であったが、E遺伝子gRNAは、3つのSARS様コロナウイルスを検出することができた（右、「E遺伝子」）。SARS-CoVおよびコウモリコロナウイルスを標的とする別個のN遺伝子gRNAは、SARS-CoV-2を検出することができなかった（中央、「N遺伝子関連種変異体」）。図107Cは、コロナウイルスDETECTRアッセイで用いられる例示的な実験機器を示す。適切なバイオセーフティ保護装置に加えて、用いられる装置は、試料回収装置、微小遠心チューブ、37°Cおよび62°Cに設定されたヒートブロック、ピペットおよびチップ、およびラテラルフローストリップを含む。図107Dは、対象におけるコロナウイルスの検出のためのDETECTRアッセイの例示的なワークフローを示す。従来のRNA抽出または試料マトリックスは、DETECTR（LAMP事前増幅およびNE遺伝子、EN遺伝子およびRNase PのCas12ベースの検出）へのインプットとして使用することができ、それは、蛍光リーダーまたはラテラルフローストリップによって可視化される。図107Eは、ラテラルフロー試験ストリップ（左）を示し、SARS-CoV-2 N遺伝子についての陽性試験結果（左、上）およびSARS-CoV-2 N遺伝子についての陰性試験結果（左、下）を示す。表（右）は、RNase P（陽性対照）、SARS-CoV-2 N遺伝子、およびコロナウイルスE遺伝子の非存在の存在を試験するラテラルフローストリップベースのコロナウイルス診断アッセイの、可能な試験指標および関連する結果を示す。
（図１０８）図108Aは、標的核酸の存在下でのCas12プログラマブルヌクレアーゼによる、FAMおよびビオチンで標識されたディテクター核酸の切断を示す（上）。ラテラルフロー試験ストリップ（下）の概略図は、試験試料中の標的核酸の存在（「陽性」）または非存在（「陰性」）のいずれかを示すマーキングを示す。完全なFAM-ビオチン化レポーター分子は、コントロール捕捉ラインに流れる。合致する標的が認識されると、Cas-gRNA複合体はレポーター分子を切断し、それは標的捕捉ラインに流れる。図108Bは、0fMのSARS-CoV-2 RNAまたは5fMのSARS-CoV-2 RNAのいずれかを含有する試料について、反応時間の効果を決定するためのLbCas12aを使用したDETECTRアッセイの結果を示す。SARS-CoV-2 N遺伝子では、RT-LAMPアンプリコンでのLbCas12a検出アッセイの蛍光信号は10分以内に飽和した。RT-LAMPアンプリコンを、2μLの5fMまたは0fMのSARS-CoV-2 N-gene IVT RNAから、62°Cで20分間増幅することによって作製した。図108Cは、図108BのDETECTRによってアッセイされた試料に相当する試料をアッセイするラテラルフロー試験ストリップを示す。0fMのSARS-CoV-2 RNA（「-」）または5fMのSARS-CoV-2 RNA（「＋」）のいずれかを含有する試料について、対照（C）または試験（T）に対応するバンドを反応時間の関数として示す。同じRT-LAMPアンプリコン上のLbCas12aは、5分以内にラテラルフローアッセイを通して可視信号を生成した。図108Dは、SARS-CoV-2の検出限界を決定するための、LbCas12a（中央）またはCDCプロトコール（左）を用いたDETECTRアッセイの結果を示す。信号は、反応当たりのウイルスゲノムのコピー数の関数として示される。アッセイの代表的なラテラルフロー結果を、0コピー/μLおよび10コピー/μLの場合について示す（右）。図108Eは、患者試料のDETECTRデータを示す。COVID-19感染症患者6人（n＝11、反復5）およびインフルエンザまたは4つの季節性コロナウイルス（HCoV-229E、HCoV-HKU1、HCoV-NL63、HCoV-OC43）のうちの1つに感染している患者12人（n＝12）からの臨床試料を、SARS-CoV-2 DETECTRを用いて分析した（明暗をつけたボックス）。ラテラルフローストリップからの信号強度をImageJを使用して定量化し、バックグラウンドを5標準偏差上回る正の閾値を用いて、N遺伝子、E遺伝子またはRNase Pセット内の最大値に正規化した。SARS-CoV-2試験の最終決定は、図107Eの解釈行列に基づいた。FluAはインフルエンザAを表し、FluBはインフルエンザBを表す。HCoVはヒトコロナウイルスを表す。図108Fは、COVID-19患者（SARS-CoV-2に対して陽性、「患者1」）、標的核酸を欠く標的なし対照試料（「NTC」）、および標的核酸を含有する陽性対照試料（「PC」）におけるSARS-CoV-2について試験するラテラルフロー試験ストリップを示す。3つすべての試料を、SARS-CoV-2 N遺伝子、SARS-CoV-2 E遺伝子、およびRNase Pの存在について試験した。図108Gは、SARS-CoV-2 DETECTRアッセイの性能特性を示す。83の臨床試料（COVID-19の陽性41、陰性42）を、SARS-CoV-2 DETECTRアッセイの蛍光バージョンを使用して評価した。1つの試料（COVID19-3）を、アッセイ品質管理の失敗により除外した。陽性コールおよび陰性コールは、図32Eに記載されている基準に基づいた。fMはフェムトモルを示し、NTCはテンプレートなしの対照を示し、PPAは陽性予測一致（positive predictive agreement）を示し、NPAは、陰性予測一致（negative predictive agreement）を示す。
（図１０９）本開示のRT-LAMPを用いたSARS-CoV-2 DETECTRアッセイと、定量的逆転写ポリメラーゼ連鎖反応（qRT-PCR）検出法を用いたSARS-CoV-2アッセイとを比較する表を示す。DETECTR RT-LAMPアッセイにおけるN遺伝子標的は、qRT-PCRアッセイで検出されたN遺伝子N2アンプリコンと同じである。
（図１１０Ａ）異なる緩衝液条件下でのRT-LAMP増幅の結果までの時間を示す。結果までの時間は、蛍光値が実験の最大値の1/3になる時間として計算した。増幅に失敗した反応は、20分での値で報告され、「no amp」と標識する。水、10％リン酸緩衝生理食塩水（PBS）、または10％ユニバーサルトランスポート培地（UTM）のいずれかにおける異なる出発濃度の標的対照プラスミドについて、結果までの時間を決定した。結果まで時間が短いほど、増幅が速いことを示す。
（図１１０Ｂ）5％ UTM、5％ PBSまたは水のいずれかにおける、SARS-CoV-2のN遺伝子、SARS-CoV-2のE遺伝子およびRNase Pの増幅効率を決定するためのRT-LAMPアッセイの結果を示す。インビトロ転写された0.5fMのN遺伝子、インビトロ転写された0.5fMのE遺伝子、および0.8ng/μLのHeLa総RNA（「N＋E＋総RNA」）を含む試料、または標的を含まない対照（「NTC」）を試験した。
（図１１０Ｃ）PBS中の鼻スワブからの直接的なRNAの増幅を示す。結果までの時間をPBS濃度の関数として測定した。鼻スワブ（「鼻スワブ」）を、HeLaトータルRNA（左、「トータルRNA:0.08ng/uL」）または水（右、「トータルRNA:0ng/uL」）のいずれかでスパイクした。鼻スワブを含まない試料（「スワブなし」）を対照として比較した。
（図１１１Ａ）SARS-CoV-2 N遺伝子（n＝6）のLbCas12a（SEQ ID NO:27）検出によって生成された生の蛍光曲線を示す。曲線は、20分未満で飽和を示した。
（図１１１Ｂ）図111Aに示す生の蛍光トレースから決定した、LbCas12aで検出されたSARS-CoV-2 N遺伝子についてのDETECTRアッセイの検出限界を示す。SARS-CoV-2 N遺伝子の濃度（コピー/mL）を減少させながら蛍光強度を測定した。
（図１１１Ｃ）図111Aに示される生の蛍光トレースから決定された、DETECTRアッセイの検出限界の結果までの時間を示す。結果まで時間が短いほど、検出が速いことを示す。
（図１１２）図112Aは、0fMのSARS-CoV-2 RNAまたは5fMのSARS-CoV-2 RNAのいずれかを含有する試料について、反応時間の効果を決定するためのLbCas12aを使用したDETECTRアッセイの結果を示す。図112Bは、図112AのDETECTRによってアッセイされた試料に相当する試料をアッセイするラテラルフロー試験ストリップを示す。0fMのSARS-CoV-2 RNA（「-」）または5fMのSARS-CoV-2 RNA（「＋」）のいずれかを含有する試料について、対照（C）または試験（T）に対応するバンドを反応時間の関数として示す。
（図１１３）異なるヒトコロナウイルス株に対するgRNAの交差反応性を決定するためのDETECTRアッセイの結果を示す。SARS-CoV-2 N遺伝子、SARS-CoV N遺伝子、bat-SL-CoVZC45 N遺伝子、SARS-CoV-2 E遺伝子、SARS-CoV E遺伝子もしくはbat-SL-CoVZC45 E遺伝子のインビトロ転写RNAを含有する試料、またはCoV-HKU1、CoV-299E、CoV-OC43もしくはCoV-NL63について陽性の臨床試料を試験した。HeLaトータルRNAをRNase Pの陽性対照として試験し、標的核酸を欠く試料（「NTC」）を陰性対照として試験した。
（図１１４）図114Aは、3つのコロナウイルス株について、N遺伝子gRNAによって標的とされる標的部位の配列アラインメント（それぞれ記載の順にSEQ ID NO：405～410）を示す。N遺伝子gRNA＃1はCDC-N2アンプリコンと適合性であり、N遺伝子gRNA＃2はWHO N-Sarbecoアンプリコンと適合性である。図114Bは、3つのコロナウイルス株について、E遺伝子gRNAによって標的とされる標的部位の配列アラインメント（それぞれ記載の順にSEQ ID NO：411～416）を示す。試験した2つのE遺伝子gRNA（E遺伝子gRNA＃1およびE遺伝子gRNA＃2）は、WHO E-Sarbecoアンプリコンと適合性である。
（図１１５）図115A～図115Cは、COVID-19感染患者試料のDETECTR動態曲線を示す。5人の患者からの10個の鼻スワブ試料（COVID19-1～COVID19-10）を、2つの異なる遺伝子、N2およびE、ならびに試料インプット対照、RNase Pを使用してSARS-CoV-2について試験した。図115Aは、標準的な増幅および検出条件を使用して、10人の患者のうち9人に、SARS-CoV-2 E遺伝子の存在を示す強い蛍光曲線が生じたことを示す（20分間の増幅、10分以内の信号）。図115Bは、10人の患者のうち8人について、SARS-CoV-2 N遺伝子が強い蛍光曲線を生成するのに必要とする増幅時間が延長されたことを示す（30分間の増幅、10分以内の信号）。図115Cは、試料インプット対照としてのRNase Pが、試験した全試料22のうち17で陽性であったことを示す（20分間の増幅、10分以内の信号）。
（図１１６）SARS-CoV-2のDETECTR分析により、約30分でウイルスゲノムが10個まで同定されることを示す（20分間の増幅、10分間のDETECTR）。LbCas12a DETECTR分析の前に、重複LAMP反応を20分間増幅した。
（図１１７）図116で提供されたLbCas12a DETECTR分析について、5分での生の蛍光を示す。SARS-CoV-2 N遺伝子の検出限界は、反応あたり10ウイルスゲノムであると決定された（n＝6）。
（図１１８）10個のCOVID-19感染患者試料および他のウイルス性呼吸器感染症についての12個の患者試料のラテラルフローDETECTR結果を示す。1つの鼻咽頭スワブ（A）および1つの中咽頭スワブ（B）を含む6人の患者からの10個の試料（COVID19-1～COVID19-5）を、2つの異なる遺伝子、N2およびE、ならびに試料インプット対照、RNase Pを使用してSARS-CoV-2について試験した。結果を図119に提供される指針に従って分析した。
（図１１９）SARS-CoV-2 DETECTRラテラルフロー結果の解釈のための規定を示す。
（図１２０Ａ）11個のCOVID-19感染患者試料および他のウイルス性呼吸器感染症の12個の患者試料の蛍光DETECTR動態曲線を示す。6人の患者からの10個の鼻咽頭/中咽頭スワブ試料（COVID19-1～COVID19-6）を、2つの異なる遺伝子、N2およびE、ならびに試料インプット対照、RNase Pを使用してSARS-CoV-2について試験した。図120Aは、標準的な増幅および検出条件を使用して試験した試料を示し、12個のCOVID-19陽性患者試料のうち10個に、SARS-CoV-2 E遺伝子の存在を示す強い蛍光曲線が生じた（20分間の増幅、10分以内の信号）。12個の他のウイルス性呼吸器臨床試料では、E遺伝子信号は検出されなかった。
（図１２０Ｂ）11個のCOVID-19感染患者試料および他のウイルス性呼吸器感染症の12個の患者試料の蛍光DETECTR動態曲線を示す。6人の患者からの10個の鼻咽頭/中咽頭スワブ試料（COVID19-1～COVID19-6）を、2つの異なる遺伝子、N2およびE、ならびに試料インプット対照、RNase Pを使用してSARS-CoV-2について試験した。図120Bは、12個のCOVID-19陽性患者試料のうち10個について、時間を延長した増幅を使用してSARS-CoV-2 N遺伝子の存在について試験された試料が、強い蛍光曲線を生成することを示す（30分間の増幅、10分以内の信号）。12個の他のウイルス性呼吸器臨床試料では、N遺伝子信号は検出されなかった。
（図１２０Ｃ）11個のCOVID-19感染患者試料および他のウイルス性呼吸器感染症の12個の患者試料の蛍光DETECTR動態曲線を示す。6人の患者からの10個の鼻咽頭/中咽頭スワブ試料（COVID19-1～COVID19-6）を、2つの異なる遺伝子、N2およびE、ならびに試料インプット対照、RNase Pを使用してSARS-CoV-2について試験した。図120Cは、試料インプット対照である、RNase Pに対応するグラフを示す。
（図１２１）示される試験解釈を用いた臨床試料についてのSARS-CoV-2 DETECTRアッセイ結果のヒートマップを示す。24個の臨床試料（COVID-19陽性、12個）を、ImageJ ゲル分析器ツールによってSARS-CoV-2 DETECTRについて定量化したラテラルフローSARS-CoV-2 DETECTRアッセイの結果（上）は、アッセイの蛍光バージョンの結果（下）と98.6％（71/72ストリップ）一致していることを示す。両方のアッセイを30分間の増幅で実行し、Cas12反応信号を10分で得た。推定陽性はオレンジ色の（＋）で示される（下、列4）。
（図１２２）示される試験解釈を用いた臨床試料についてのSARS-CoV-2 DETECTRアッセイ結果のヒートマップを示す。上のプロットは、追加の30個のCOVID-19陽性臨床試料（陽性27、推定陽性1、陰性2）に対する蛍光SARS-CoV-2 DETECTRアッセイの結果を示す。推定陽性はオレンジ色の（＋）で示される（上、列9）。下のプロットは、追加の30個のCOVID-19陰性臨床試料（陽性0、陰性30）に対する蛍光SARS-CoV-2 DETECTRアッセイの結果を示す。
（図１２３）異なるプライマーセットを用いたRNase P POP7のRT-LAMP増幅について、結果までの時間を示す。プライマーセット1～10を用いて増幅した試料について、結果までの時間を決定した。プライマーセット1は、SEQ ID NO:360-SEQ ID NO:365に対応し、プライマーセット9は、SEQ ID NO:366-SEQ ID NO:371に対応する。
（図１２４）プライマーセット1またはプライマーセット9を用いてRT-LAMPを使用して増幅され、R779、R780またはR1965 gRNAで検出されたRNase P POP7に行われたDETECTR反応の経時的な生蛍光を示す。DETECTR反応は、37°Cで90分間行った。セット1プライマーによって生成されたアンプリコンは、R779によってバックグラウンドなし（点線）で検出された。
（図１２５）図125Aは、プライマーセット1またはプライマーセット9を用いるRT-LAMPを使用して増幅されたトータルRNAの10倍希釈物を含む試料における、RNase P POP7検出の結果までの時間を示す。増幅は、60°Cで30分間行った。図125Bは、図125Aに示され、gRNA 779（SEQ ID NO:330）またはgRNA 1965（SEQ ID NO:331）を使用して検出されたRNase P POP7アンプリコンのDETECTR反応を示す。プライマーセット1を使用して増幅した試料をgRNA 779で検出し、プライマーセット9を使用して増幅した試料をgRNA 1965で検出した。DETECTR反応は、37°Cで90分間行った。
（図１２６）図126Aおよび図126Bは、DETECTRアッセイで使用するために設計されたカートリッジの写真を示す。
（図１２７）図127Aおよび図127Bは、図126Aの写真で示されるカートリッジの概略図である。
（図１２８Ａ）DETECTRアッセイで使用するために設計されたカートリッジの概略図を示す。図128Aは、円形の試薬貯蔵ウェルおよびz方向の高抵抗蛇行経路を有するカートリッジを示す。
（図１２８Ｂ）DETECTRアッセイで使用するために設計されたカートリッジの概略図を示す。図128Bは、細長い試薬貯蔵ウェルおよびz方向の高抵抗蛇行経路を有するカートリッジを示す。
（図１２８Ｃ）DETECTRアッセイで使用するために設計されたカートリッジの概略図を示す。図128Cは、円形の試薬貯蔵ウェルおよびxy方向の高抵抗蛇行経路を有するカートリッジを示す。
（図１２８Ｄ）DETECTRアッセイで使用するために設計されたカートリッジの概略図を示す。図128Dは、細長い試薬貯蔵ウェルおよびxy方向の高抵抗蛇行経路を有するカートリッジを示す。
（図１２９）図129A～図129Dは、DETECTRアッセイで使用するために設計されたカートリッジの概略図を示す。図129Aは、試料計量チャネルとは異なる平面または層上で蛇行している、試料計量のための蛇行抵抗チャネルを有するカートリッジを示す。図129Bは、試料計量チャネルと同じ平面または層上で蛇行している、試料計量のための蛇行抵抗チャネルを有するカートリッジを示す。図129Cは、直角の困難な経路（arduous path）の試料計量のための抵抗性経路、および試料計量チャネルとは異なる平面または層上のDETECTR試料計量投入口を含むカートリッジを示す。図129Dは、試料計量のための直角の困難な経路抵抗性経路、および試料計量チャネルと同じ平面または層上のDETECTR試料計量投入口を含むカートリッジを示す。
（図１３０）図130Aは、DETECTRアッセイで使用するために設計されたカートリッジの特徴を示す。図130Bは、本開示のカートリッジを製造するための製造スキーム（左および中央）およびカートリッジ内の試料を検出するための読み出し装置（右）を示す。
（図１３１）図131Aは、本開示のカートリッジの領域を加熱するためのカートリッジマニホールドの概略図を示す。カートリッジマニホールドは、一体化された空気供給接続部および空気供給インターフェース用の一体化されたOリング溝を備える、一体化された加熱ゾーンを有する。カートリッジマニホールドは、増幅温度ゾーンを検出温度ゾーンから熱的に分離し、カートリッジの増幅チャンバおよび検出チャンバの適切な温度を維持するための絶縁ゾーンを含む。図131Bは、本明細書に記載のカートリッジを製造するための2つの製造方法を示す。第1の製造方法（左）では、多層の二次元（2D）積層を用いてカートリッジを製造する。第2の製造方法（右）では、統合された複雑な形質を含む部品が射出成形され、ラミネーションによって封止される。図131Cは、カートリッジをシリンジに結合するためのルアースリップアダプタを備えるカートリッジの概略図を示す。アダプタは、スリップルアーチップを有するしまりばめ封止を形成することができる。アダプタは、本明細書に開示されるカートリッジのいずれかと共に機能するように構成される。
（図１３２）図132Aおよび図132Bは、マイクロ流体カートリッジと共に使用するための一体化フローセルの概略図を示す。一体化フローセルは、溶解領域、増幅領域、および検出領域の3つの領域を含む。溶解領域は、マイクロ流体チップショップ試料溶解フローセルを収容するのに十分な長さである。溶解フローセルは、本明細書に開示されるカートリッジ上の増幅および検出チャンバと組み合わせてもよい。
（図１３３）本開示のカートリッジ上の投入口チャネルの詳細を示す。
（図１３４）本開示のマイクロ流体カートリッジを使用してDETECTRアッセイを実行するためのワークフローを示す。カートリッジ（「チップ」）に試料および反応溶液を充填する。増幅チャンバ（「LAMPチャンバ」）を60°Cに加熱し、試料を増幅チャンバ内で30分間インキュベートする。増幅された試料（「LAMPアンプリコン」）が、DETECTR反応チャンバにポンプで圧送され、DETECTR試薬が、DETECTR反応チャンバにポンプで圧送される。DETECTR反応チャンバを37°Cに加熱し、試料を30分間インキュベートする。DETECTR反応チャンバ内の蛍光をリアルタイムで測定して、定量的結果を生成する。
（図１３５）本明細書に開示されるカートリッジと互換性のあるカートリッジマニホールドのシステム電子機器の構造の概略図を示す。電子機器は、カートリッジの第1のゾーンを37°Cに、カートリッジの第2のゾーンを60°Cに加熱するように構成されている。
（図１３６）図136Aおよび図136Bは、本開示のカートリッジを加熱および検出するためのカートリッジマニホールドの概略図を示す。マニホールドは、カートリッジを受け入れ、DETECTR反応を促進し、DETECTR反応の結果生じる蛍光を読み取るように構成される。
（図１３７Ａ）カートリッジ内の蛍光試料の一例を示し、カートリッジマニホールドで照射される。陽性対照ウェルは、60°Cで30分間の増幅工程および37°Cで30分間の検出工程後の試薬および増幅試料を含有する。空のウェルは、偽陰性試料として機能する。
（図１３７Ｂ）本開示のカートリッジを加熱および検出するためのカートリッジマニホールドを示す。
（図１３７Ｃ）本開示のカートリッジを加熱および検出するためのカートリッジマニホールドを示す。
（図１３８）図138Aおよび図138Bは、本開示のマニホールドによって促進されるマイクロ流体カートリッジの検出チャンバ内で生成される蛍光を示す。
（図１３９）図139Aおよび図139Bは、60°Cに加熱された増幅チャンバ（図139A）または37°Cに加熱されたDETECTRチャンバ（図139B）の熱試験概要を示す。
（図１４０）図140Aおよび図140Bは、60°Cに加熱された増幅チャンバ（図140A）または37°Cに加熱されたDETECTRチャンバ（図140B）の熱試験概要を示す。
（図１４１）図141Aは、マイクロ流体カートリッジからのLAMP生成物をインプットとして使用して、ゲイン100でプレートリーダー上で実行されたDETECTR結果を示す。試料は、単一の非テンプレート対照（NTC）と共に2つ組で実行された。図141Bは、マイクロ流体チップからの試料を使用してプレートリーダー上で実施される3つのLAMP生成物を示す。LAMP反応は、チップが実行された順序で番号付けされる（最初に実行されたLAMP＿1など）。ドナーはSNP Aについてホモ接合性であり、それによれば、crRNA 570が最初に現れる。ATTO 488を蛍光標準として使用した。
（図１４２）図142Aは、充填されたマイクロ流体チップの画像を示す。図142Bは、30分間のLAMP増幅後にプレートリーダーで測定したDETECTR反応の結果を示す。
（図１４３）図143A、図143B、図143Cおよび図143Dは、コロナウイルスDETECTR反応の結果を示す。10コピーがLAMPにインプットされた2つの反応チャンバは、DETECTR信号の急速な増加をもたらした。すべてのNTCは陰性であった。10コピーがLAMPに入力されると、以下の図143Cのフォトダイオード測定値に示されるように、DETECTR信号は反応の過程にわたって徐々に増加した。図143Dの陰性対照は、汚染がないことを示した。
（図１４４）図144A、図144B、図144Cおよび図144Dは、反復コロナウイルスDETECTR反応の結果を示す。
（図１４５）図145Aおよび図145Bは、マイクロ流体カートリッジにおけるインフルエンザB DETECTR反応のフォトダイオード測定値を示す。
（図１４６）図146A、図146Bおよび図146Cは、マイクロ流体カートリッジにおけるインフルエンザB DETECTR反応のフォトダイオード測定値を示す。
（図１４７）ガラスキャピラリーに7ヶ月間保存された一連のDETECTR試薬からの蛍光結果を示す。
（図１４８）スピンスルーカラム（spin-through column）の設計、ならびに連続増幅およびDETECTR反応のためのスピンスルーカラムの使用方法を提供する。
（図１４９）電気化学的に検出可能な核酸を構築するために使用される3つの試薬:（A）フェロセン標識チミジン、（B）6-カルボキシフルオレセイン、および（C）ビオチン標識ホスフェートの構造を提供する。
（図１５０）試料インプットチャンバと、試料の一部を増幅およびディテクター反応に供することができる複数のチャンバとを含む射出成形カートリッジの設計を提供する。
（図１５１）図150に示す射出成形カートリッジを用いることができる、検出器ダイオードアレイおよび加熱パネルを含む装置の設計を提供する。
（図１５２）異なる二重溶解増幅緩衝液に供した試料で実施した一連のDETECTR反応からの蛍光データを示す。
（図１５３）異なる二重溶解増幅緩衝液に供した試料で実施した一連のDETECTR反応からの蛍光データを示す。
（図１５４）パネル（a）は、試料に複数の増幅およびDETECTR反応を実施するための射出成形カートリッジの設計を提供する。パネル（b）は、射出成形カートリッジを用い、カートリッジで行われるDETECTR反応からの蛍光を測定するように構成された装置の設計を提供する。
（図１５５）試料に並列増幅およびDETECTR反応を実行するために、射出成形カートリッジ、および図154に示す装置を用いる方法を提供する。
（図１５６）射出成形カートリッジおよび図154の装置からのダイオードアレイおよび色素充填反応区画を示す。
（図１５７）5つの増幅チャンバに接続された1つの試料チャンバと、各増幅チャンバに接続された2つの検出チャンバとを含む射出成形カートリッジの可能な設計を示す。したがって、装置は、単一の試料に対して10の並行したDETECTR反応を実行することができる。
（図１５８）4つの増幅チャンバに接続された1つの試料チャンバと、各増幅チャンバに接続された2つの検出チャンバとを含む射出成形カートリッジの可能な設計を示す。射出成形カートリッジは、カートリッジ全体の流れを制御するポンプマニホールドを圧送するための、一連のバルブおよびポンプまたはポートを含む。
（図１５９）4つの増幅チャンバに接続された1つの試料チャンバと、各増幅チャンバに接続された2つの検出チャンバと、試料チャンバに接触された試薬チャンバとを含む射出成形カートリッジの可能な設計を示す。
（図１６０）増幅および検出チャンバに通じる流路内に試薬チャンバを有する射出成形カートリッジ設計の上面図を提供する。
（図１６１）単一の回転バルブによって複数の試薬および増幅チャンバに接続することができる試料チャンバを有する、射出成形カートリッジ設計の一部を示す。
（図１６２）複数の区画を接続するスライドバルブを有する射出成形カートリッジ設計の一部を示す。パネルA～Cは、スライドバルブが選ぶことができる様々な位置を示す。
（図１６３）パネルAは、ケーシングを有する射出成形カートリッジの可能な設計を示す。パネルBは、パネルAに示されている設計の物理モデルを提供する。
（図１６４）パネルAは、ケーシングを有する射出成形カートリッジの設計の底面図を提供する。パネルBは、射出成形カートリッジの上部の図を提供する。
（図１６５）スライドバルブを有する射出成形カートリッジの複数の図を提供する。
（図１６６）透明な反応チャンバにつながる複数の試薬ウェルを有する射出成形カートリッジの一部の図を2つ提供する。
（図１６７）パネルA～Bは、射出成形カートリッジ設計の上面図を提供する。パネルCは、射出成形カートリッジの物理モデルの写真を示す。
（図１６８）ダイオードアレイを含む装置に収容された射出成形カートリッジの写真を示す。
（図１６９）射出成形カートリッジおよび検出用のダイオードアレイを含む装置を制御するためのグラフィックユーザインターフェースを示す。
（図１７０）8ダイオード検出器アレイ、8チャンバ射出成形カートリッジ、および色素を用いた一連の蛍光実験の結果を示す。
（図１７１）8ダイオード検出器アレイを用いて測定された、一連のHERC2ターゲティングDETECTR反応および緩衝液対照からの蛍光結果を示す。
（図１７２）DETECTR反応を含有する8つのチャンバと共に、装置に挿入された射出成形カートリッジを示す。
（図１７３）異なる溶解条件下でLAMPを使用したSeraCare標的核酸の増幅の結果を示す。緩衝液（上のプロット）またはウイルス溶解緩衝液（「VLB」、下のプロット）のいずれかを含有する低pH緩衝液中で試料を増幅した。緩衝剤は、還元剤を含まないか（「制御」、列1および4）、還元剤Bを含むか（列2および5）、または還元剤Aを含んだ（列3および6）。試料を室温（左のプロット）または95°C（右のプロット）のいずれかで5分間インキュベートした。試料は、標的を含まない（「NTC」）か、反応あたり2.5、25、または250コピーのいずれかを含む。25μLの反応物において5μLの試料を使用してアッセイを3つ組で行った。
（図１７４）異なる溶解条件下でLAMPを使用したSeraCare標準標的核酸の増幅の結果を示す。緩衝液（左のプロット）またはウイルス溶解緩衝液（「VLB」、右のプロット）のいずれかを含有する低pH緩衝液中で試料を増幅した。緩衝液は、還元剤を含まない（「対照」）か、還元剤B、または還元剤Aを含んだ。試料を室温（上のプロット）または95°C（下のプロット）のいずれかで5分間インキュベートした。試料は、標的を含まない（「NTC」）か、反応あたり1.5、2.5、15、25、150、または250コピーのいずれかを含む。15μLの反応物において3μLの試料、または25μLの反応物において5μLの試料を使用してアッセイを3つ組で実施した。
（図１７５）6つの異なるウイルス溶解緩衝液（「VLB」、「VLB-D」、「VLB-T」、「緩衝液」、「緩衝液A」、「緩衝液B」）の存在下での、SARS-CoV-2 N遺伝子（「N」）およびRNase P試料インプット対照核酸（「RP」）の増幅を示す。緩衝液-Aは還元剤Aを含む緩衝液を含有し、緩衝液-Bは還元剤Bを含む緩衝液を含有する。陰影付きの正方形は増幅速度を示し、陰影が濃いほど速い増幅を示す。増幅を、力価の高い、中力価または低力価のCOVID-19陽性患者試料（それぞれ「16.9」、「30.5」、「33.6」）に対して95°C（「95 C」）または室温（「RT」）で行った。試料は2つ組で測定した。
（図１７６）電気活性レポーター核酸を用いて実施したDETECTR反応の矩形波ボルタンメトリー結果を示す。結果は、DETECTR反応の開始直後（0分）および33分後に収集した。
（図１７７）電気活性レポーター核酸を用いて実施したDETECTR反応のサイクリックボルタンメトリー結果を示す。結果は、DETECTR反応の開始直後（0分）および26分後に収集した。
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure may be had by reference to the following detailed description and accompanying drawings, which set forth illustrative aspects in which the principles of the disclosure are employed.
(FIG. 1) Schematic showing the workflow of the CRISPR-Cas reaction. Step 1 shown in the workflow is sample preparation, and Step 2 shown in the workflow is nucleic acid amplification. Step 3 shown in the workflow is the Cas reaction incubation. Step 4 shown in the workflow is detection (readout). Non-essential steps are indicated by oval circles. If detection and readout are incorporated into the CRISPR reaction, steps 1 and 2 are not essential and steps 3 and 4 can be performed simultaneously.
FIG. 2 shows an exemplary fluidic apparatus for sample preparation that may be used in step 1 of the workflow schematic of FIG. The sample preparation fluidics device shown in this figure can process different types of biological samples: swabs containing fingerstick blood, urine, or faeces, buccal or other collections.
(FIG. 3) Three exemplary fluidic devices for Cas reactions, including fluorescence or electrochemical readout, that can be used in steps 2-4 of the workflow schematic of FIG. This figure shows the device performing three iterations of steps 2-4 of the workflow schematic of FIG.
FIG. 4 shows schematics of readout processes that may be used, including (a) fluorescence readout and (b) electrochemical readout.
(FIG. 5) Exemplary fluidic setup for coupled invertase/Cas reaction, including colorimetric or electrochemical/glucometer readout. This figure shows a fluidic device for minimizing the Cas reaction coupled with the enzyme invertase. The readout process, which includes surface modification and (a) optical readout using DNS or other compounds, and (b) electrochemical readout (electrochemical analyzer or blood glucose meter), is shown in the exploded view scheme at the bottom. shown in
(FIG. 6) FIG. 6A shows a panel of gRNAs for RSV evaluated for detection efficiency. Darker squares in a background subtracted row indicate higher efficiency in detecting RSV target nucleic acids. FIG. 6B shows a graph of gRNA pools against background-subtracted fluorescence.
(FIG. 7) Shows individual components of the sample preparation device of the present disclosure.
(FIG. 8) A sample workflow using the sample processing device.
(FIG. 9) shows the extraction buffer used to extract influenza A RNA from the rest of the clinical samples.
(Fig. 10) Low pH conditions enable rapid extraction of influenza A genomic RNA.
(FIG. 11) Application of RT-RPA to the detection of viral RNA of influenza A, influenza B, and human respiratory syncytial virus (RSV) by Cas12a. The schematic on the left shows the workflow including DNA/RNA, RPA/RT-RPA provision, and Cas12a detection. The graph on the right shows the results of Cas12a detection as measured by fluorescence over time.
(FIG. 12) shows the application of RT-RPA in combination with the IVT reaction, allowing detection of viral RNA using Cas13a. The schematic on the left shows the workflow including DNA/RNA, RPA/RT-RPA provision, in vitro transcription, and Cas13a detection. The graph on the right shows the results of Cas13a detection as measured by fluorescence for each test condition.
(FIG. 13) RNA production from RNA viruses using the RT-RPA-IVT 'two-pot' reaction, detected by Casl3a. The schematic on the left shows the workflow, including DNA/RNA provision, a "two-pot" reaction including RPA/RT-RPA and in vitro transcription in the first reaction, and Cas13a detection in the second reaction. The graph on the right shows the results of Cas13a detection as measured by fluorescence for each test condition.
(FIG. 14) Shows the effect of various buffers on the performance of the one-pot Cas13a assay. The schematic on the left shows the workflow including DNA/RNA and RPA/RT-RPA provision, in vitro transcription, and Cas13a detection. The graph on the right shows the results of Cas13a detection as measured by fluorescence for each test condition.
FIG. 15. Specific detection of viral RNA from Peste des petits ruminants (PPR) virus infecting goats using a one-pot Cas13a assay. The schematic on the left shows the workflow including DNA/RNA and RPA/RT-RPA provision, in vitro transcription, and Cas13a detection. The graph on the right shows the results for the conditions tested, Cas13a detection measured by fluorescence over time.
(Fig. 16) Specific detection of influenza B using a one-pot Cas13a assay performed at 40°C. 40 fM of viral RNA was added to the reaction. The schematic on the left shows the workflow including DNA/RNA and RPA/RT-RPA provision, in vitro transcription, and Cas13a detection. The graph on the right shows the results of Cas13a detection as measured by fluorescence for each test condition.
(FIG. 17) Shows the resistance of a one-pot Cas13a assay for detection of RNA from influenza B virus at 40° C. in the presence and absence of a universal transport medium called universal transport medium (UTM Copan). The schematic on the left shows the workflow including DNA/RNA and RPA/RT-RPA provision, in vitro transcription, and Cas13a detection. The graph on the right shows the results for each test condition of Cas13a detection as measured by fluorescence over time.
(FIG. 18) One-pot Cas13a detection assay at various temperatures. FIG. 18A shows a schematic of the workflow including DNA/RNA provision and a one-pot reaction including RPA/RT-RPA, in vitro transcription, and Cas13a detection. Figure 18B shows a graph of Cas13a detection of influenza A RNA at various temperatures. FIG. 18C shows a graph of Cas13a detection of influenza B RNA at various temperatures. Figure 18D shows a graph of Cas13a detection of human RSV RNA at various temperatures.
FIG. 19. Optimization of LAMP reactions for detection of internal amplification controls using DNA sequences from Mammuthus primigenius (woolly mammoth) mitochondria. Figure 19A shows a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, and Cas12a detection. FIG. 19B shows the time at which the LAMP reaction occurs for an internal amplification control using a DNA sequence from Mammusus primigenius, quantified by fluorescence. Figure 19C shows Casl2a-specific detection of LAMP amplicons at 37°C from a 68°C temperature response.
(FIG. 20) LAMP optimization and Cas12-specific detection of the human POP7 gene. The human POP7 gene is an RNase P

is a component of FIG. 20A shows a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, and Cas12a detection. FIG. 20B shows the time to outcome of the LAMP/RT-LAMP reaction for RNase P POP7 at different temperatures, quantified by fluorescence. FIG. 20C shows three graphs showing Cas12a-specific detection at 37° C. of LAMP/RT-LAMP amplicons from a 68° C. temperature response.
(FIG. 21) Specific detection of three different RT-LAMP amplicons for influenza A virus. On the left is a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, and Cas12a detection. On the right is a graph showing the results for each test condition of Cas12a detection measured by fluorescence over time.
(FIG. 22) Identification of optimal crRNAs for specific detection of influenza B (IBV) RT-LAMP amplicons. On the left is a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, and Cas12a detection. On the right is a graph showing the results of Cas12a detection measured by fluorescence over time for each test condition (IAV is influenza A virus, IBV is influenza B virus, NTC is no template control).
FIG. 23 shows the results of a 1% agarose gel with bands representing products of the RT-LAMP reaction.
(FIG. 24) Cas12a discrimination between amplicons from multiplexed RT-LAMP reactions for Influenza A and Influenza B. FIG. FIG. 24A shows a schematic of a workflow including viral RNA, multiplexed RT-LAMP provision, and Cas12a influenza A or Cas12a influenza B detection. FIG. 24B shows Cas12a detection of RT-LAMP amplicons after multiplexed RT-LAMP amplification at 60° C. for 30 minutes. FIG. 24C shows background-subtracted fluorescence of Cas12a detection of RT-LAMP amplicons for 10,000 viral genome copies of IAV and IBV at 37° C. for 30 minutes.
(FIG. 25) Between triplicate multiplexed RT-LAMP reactions for Influenza A, Influenza B, and Mammousus primigenius (woolly mammoth) mitochondrial internal amplification control sequences after multiplexed RT-LAMP amplification for 30 minutes at 60°C. Cas12a identification is shown. At the top is a schematic of the workflow including viral RNA, multiplexed RT-LAMP provision, and Cas12a influenza A detection or Cas12a influenza B detection or Cas12 internal amplification control detection. Below is a graph showing the results for each test condition of Cas12 detection measured by fluorescence over time.
FIG. 26 shows a schematic of LAMP and RT-LAMP primer design. Figure 26A shows a schematic showing the identity of the primers used in LAMP and RT-LAMP. Primers LF and LB are optional in some LAMP and RT-LAMP designs, but generally increase the efficiency of the reaction. Figure 26B shows a schematic showing the position and orientation of the T7 promoter in various LAMP primers.
(FIG. 27) shows that the T7 promoter can be included in the F3 or B3 primers (outer primers), or the FIP or BIP primers for Influenza A. FIG. 27A shows a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, in vitro transcription, and Cas13a detection. FIG. 27B shows the time to result of the influenza A RT-LAMP reaction using different primer sets, quantified by fluorescence. Figure 27C shows in vitro transcription (IVT) by T7 RNA polymerase of the products of the RT-LAMP reaction for influenza A using different primer sets at 37°C for 10 minutes.
(FIG. 28) Detection of RT-SIBA amplicon for Influenza A by Cas12. On the left is a schematic of the workflow including DNA/RNA, SIBA/RT-SIBA provision, and Cas12a detection. On the right is a graph showing Cas12a detection measured by fluorescence for each of the conditions tested.
(FIG. 29) Layout of Milenia commercial strips with typical reporters.
(FIG. 30) Layout of Milenia HybridDetect 1 strip, including amplicons.
(FIG. 31) Shows the layout of a Milenia HybridDetect 1 strip containing a standard Cas reporter.
(Fig. 32) A modified Cas reporter containing a DNA linker to biotin-dT (indicated by pink hexagons) bound to a FAM molecule (indicated by green starts).
(FIG. 33) Layout of Milenia HybridDetect strips containing engineered Cas reporters.
(FIG. 34) An example of a single target assay format (left) and a multiplexed assay format (right).
(FIG. 35) Another variation of an assay before use (top), with a positive result (left middle), with a negative result (right middle), and a failed test (bottom).
(FIG. 36) One design of a tethered lateral flow Cas reporter.
(FIG. 37) Shows the workflow for CRISPR diagnostics using tethered cleavage reporters using magnetic beads.
(FIG. 38) Shows a schematic of an enzyme-reporter system that is filtered by streptavidin-biotin before reaching the reaction chamber.
(FIG. 39) Invertase nucleic acid used for detection of target nucleic acid. The invertase-nucleic acid is immobilized on magnetic beads and added to a sample reaction containing Cas protein, guide RNA and target nucleic acid. Target recognition activates the Cas protein to cleave the invertase-nucleic acid, releasing the invertase enzyme from the immobilized magnetic beads. This solution contains sucrose and DNS reagent and is transferred to a "reaction mixture" that changes color from yellow to red as invertase converts sucrose to glucose or to a handheld glucose meter device for digital readout. be able to.
(FIG. 40) Shows the layout of one of the two-pot DETECTR assays. In this layout, a swab collection cap seals the swab reservoir chamber. Clockwise to the swab reservoir chamber is a chamber holding the amplification reaction mixture. Clockwise to the chamber holding the amplification reaction mixture is the chamber holding the DETECTR reaction mixture. Clockwise to this is the detection area. Clockwise to the detection area is the pH balance well. Cartridge well caps are shown, sealing all wells containing various reagent mixtures. The cartridges themselves are shown as square layers at the bottom of the schematic. On the right is shown a diagram of the instrument piper pump that drives the fluid in each chamber/well and is connected throughout the cartridge. Below the cartridge is a rotary valve that connects to the instrument.
FIG. 41 shows one workflow of various reactions in the two-pot DETECTR assay of FIG. First, a swab can be inserted into a 200ul swab chamber and mixed as shown in the upper left figure. In the middle left figure, rotate the valve clockwise to the "swab chamber position" and take a 1 uL sample. In the lower left figure, rotate the valve clockwise to the "Amplification Reaction Mix" position and dispense 1 ul of sample to mix. In the upper right figure, 2uL of sample is aspirated from the "Amplification Reaction Mix". In the top middle view, the valve is rotated clockwise to the "DETECTR" position, the sample is dispensed and mixed, and 20ul of sample is aspirated. Finally, in the lower right figure, rotate the valve clockwise to the detection area position and dispense 20 ul of sample.
(FIG. 42) Shows a variation of the workflow shown in FIG. 41, which is also consistent with the method and system of the present disclosure. The left side is the view shown in the upper right of FIG. On the right is a modified view, where there is a first amplification chamber counterclockwise to the swab lysis chamber and a second amplification chamber clockwise to the swab lysis chamber. In addition, clockwise to Amplification Chamber #2, there are two sets of "Dual" DETECTR chambers labeled "Dual DETECTR Chamber #2" and "Dual DETECTR Chamber #1" respectively.
(FIG. 43) A breakdown of the workflow for the modified layout shown in FIG. Specifically, from a swab lysis chamber holding 200 ul of sample, 20 ul of sample can be transferred to amplification chamber #1 and 20 ul of sample can be transferred to amplification chamber #2. After amplification in Amplification Chamber #1, 20 ul of sample can be transferred to Dual DETECTR Chamber #1a and 20 ul of sample can be transferred to Dual DETECTR Chamber #1b. Additionally, after amplification in amplification chamber #2, 20 ul of sample can be transferred to dual DETECTR chamber #2a and 20 ul of sample can be transferred to dual DETECTR chamber #2b.
(FIG. 44) A modification of the cartridge shown in FIGS. 43 and 42 is shown.
45 shows a top view of the cartridge of FIG. 44; FIG. This layout and workflow has a reproduction to compare with the layout and workflow of Figures 40-41.
(Fig. 46) Shows the layout of the two-pot DETECTR assay. At the top is shown a pneumatic pump that connects with the cartridge. In the center is shown a top view of the cartridge showing the top layer with the reservoir. At the bottom is shown a slide valve containing the sample and an arrow pointing to the lysis chamber on the left, followed by the amplification chamber on the right and the DETECT chamber on the right.
FIG. 47 shows a comparison of the DETECTR assay disclosed herein to the gold standard PCR-based method of detecting target nucleic acids. A flow chart is shown showing the gradient of the sample preparation evaluation from crude (left) to pure (right). Sample preparation steps that convert a crude sample into a pure sample include lysing, binding, washing and eluting. The DETECTR assay disclosed herein requires only the sample preparation step of lysis and can result in crude samples. PCR-based methods, on the other hand, require lysis, binding, washing and elution and can result in very pure samples.
(FIG. 48) Cas13a detection of target RT-LAMP DNA amplicons. Figure 48A shows a schematic of the workflow including DNA/RNA, LAMP/RT-LAMP provision, and Cas13a detection. FIG. 48B shows Cas13a-specific detection of target RT-LAMP DNA amplicons containing the first primer set as measured by background-subtracted fluorescence on the y-axis. FIG. 48C shows Cas13a-specific detection of target RT-LAMP DNA amplicons containing the second primer set as measured by background-subtracted fluorescence on the y-axis.
(FIG. 49) FIG. 49A shows a Casl3 detection assay using 2.5 nM RNA, single-stranded DNA (ssDNA) or double-stranded (dsDNA) as the target nucleic acid, with detection by fluorescence for each of the target nucleic acids tested. It was measured. FIG. 48B shows a Cas12 detection assay using 2.5 nM RNA, ssDNA and dsDNA as target nucleic acids, with detection measured by fluorescence for each of the target nucleic acids tested. FIG. 48C shows the performance of Cas13 and Cas12 against various concentrations of target RNA, target ssDNA and target dsDNA, detection was measured by fluorescence for each of the target nucleic acids tested.
(FIG. 50) LbuCasl3a detection assay using 2.5 nM target ssDNA with 170 nM of various reporter substrates, detection was measured by fluorescence for each of the reporter substrates tested.
(FIG. 51) FIG. 51A shows the results of Cas13 detection assays for LbuCasl3a (SEQ ID NO:131) and LwaCasl3a (SEQ ID NO:137) using 10 nM or 0 nM of target RNA. Detection was measured over time by fluorescence resulting from reporter cleavage. FIG. 51B shows the results of Casl3 detection assays for LbuCasl3a (SEQ ID NO:131) and LwaCasl3a (SEQ ID NO:137) using 10 nM or 0 nM target ssDNA. Detection was measured over time by fluorescence resulting from reporter cleavage.
(FIG. 52) LbuCasl3a (SEQ ID NO:131) detection assay using 1 nM target RNA (left) or target ssDNA (right) in buffers with various pH values ranging from 6.8 to 8.2.
(FIG. 53) FIG. 53A shows guide RNA (gRNA) tiled along the target sequence at 1-nucleotide intervals. Figure 53B shows the LbuCasl3a (SEQ ID NO:131) detection assay using 0.1 nM RNA or 2 nM target ssDNA containing gRNAs tiled at 1-nucleotide intervals and off-target gRNAs. Figure 53C shows the data from Figure 97B ranked by target ssDNA performance. Figure 53D shows gRNA performance for each nucleotide on the 3' end of the target RNA. Figure 53E shows gRNA performance for each nucleotide on the 3' end of the target ssDNA.
(FIG. 54) FIG. 54A shows an LbuCasl3a detection assay using 1 μL of target DNA amplicon from various LAMP isothermal nucleic acid amplification reactions. Figure 54B shows an LbuCas13a (SEQ ID NO:131) detection assay using various amounts of PCR reactions as target DNA.
FIG. 55 shows the pneumatic valve apparatus layout for the DETECTR assay. FIG. 55A shows a schematic diagram of a pneumatic valve device. A pipette pump aspirates and dispenses the sample. Connect the air manifold to the pneumatic pump to open and close the normally closed valves. Pneumatic devices move fluid from one location to the next. The pneumatic design has reduced channel crosstalk compared to other device designs. Figure 55B shows a schematic of a cartridge for use with the oscillating valve pneumatic device shown in Figure 55A. A valve configuration is shown. A normally closed valve (one such valve is indicated by an arrow) has an elastomeric seal at the top of the channel to isolate each chamber from the rest of the system when the chamber is not in use. A pneumatic pump uses air to open and close valves as needed to move fluid to the required chambers within the cartridge.
56 shows a valve circuit layout of the pneumatic valve device shown in FIG. 55A; FIG. Place the sample in the sample well with all valves closed as shown in (i). Dissolve the sample in the sample well. The lysed sample is transferred from the sample chamber to the second chamber by opening the first vibrating valve and aspirating the sample using a pipette pump, as shown in (ii). The sample is then transferred to the first amplification chamber by closing the first oscillating valve and opening the second oscillating valve, as shown in (iii), where it is mixed with the amplification mixture. After the sample is mixed with the amplification mixture, it is moved to the next chamber by closing the second oscillating valve and opening the third oscillating valve, as shown in (iv). The sample is moved to the DETECTR chamber by closing the third oscillating valve and opening the fourth oscillating valve, as shown in (v). The sample can be moved through different series of chambers by opening and closing different series of normally open (eg, vibrating type) valves, as shown in (vi). Actuation of individual valves within a desired series of chambers prevents cross-contamination between channels.
(FIG. 57) Shows a schematic diagram of a slide valve device. The offset pitch of the channels allows for aspiration and dispensing into each well separately and helps mitigate cross-talk between amplification chambers and corresponding chambers.
58 shows a diagram of sample movement through the slide valve device shown in FIG. 57. FIG. In the initial closed position (i), sample is loaded into the sample well and dissolved. A slide valve is then actuated by the instrument and sample is loaded into each channel using a pipette pump that dispenses the appropriate amount into the channel (ii). Samples are delivered to the amplification chamber by actuating a slide valve and mixed with a pipette pump (iii). Samples from the amplification chambers are aspirated into each channel (iv) and then dispensed into each DETECTR chamber and mixed (v) by actuating slide valves and pipette pumps.
FIG. 59 shows a schematic view of the top layer of the cartridge of the pneumatic valve device of the present disclosure, with appropriate dimensions highlighted. The schematic shows one 2 inch by 1.5 inch cartridge.
FIG. 60 shows a schematic diagram of a modified top layer of a cartridge of the pneumatic valve device of the present disclosure, adapted to electrochemical dimensions; In this schematic, three lines are shown in the detection chambers (the four rightmost chambers). These three lines represent wires (or "metal leads") that are co-molded, 3D printed, or hand-assembled into the disposable cartridge to form a three-electrode system.
FIG. 61 shows a scheme for designing primers for loop-mediated isothermal amplification (LAMP) of target nucleic acid sequences. Regions labeled with a "c" are reverse complementary to corresponding regions without a "c" (eg, region F3c is reverse complementary to region F3).
(FIG. 62) Nucleic acid sequences corresponding to or annealing to LAMP primers or guide RNA sequences or containing protospacer adjacent motifs (PAM) or protospacer adjacent sites (PFS) and amplification and detection by LAMP and DETECTR 1 shows schematic representations of exemplary configurations of various regions of a target nucleic acid sequence for . Figure 62A shows a schematic representation of exemplary placement of guide RNAs (gRNAs) relative to various regions of the nucleic acid sequences that correspond to or anneal to LAMP primers. In this arrangement, the guide RNA is reverse complementary to the sequence of the target nucleic acid that lies between the F1c region (ie, the region that is reverse complementary to the F1 region) and the B1 region. Figure 62B shows a schematic representation of exemplary placement of guide RNA sequences relative to various regions of the nucleic acid sequences that correspond to or anneal to LAMP primers. In this arrangement, the guide RNA is partially reverse complementary to the sequence of the target nucleic acid that lies between the F1c and B1 regions. For example, the target nucleic acid includes sequences between the F1c and B1 regions that are reverse complementary to at least 60% of the guide nucleic acid. In this arrangement the guide RNA is not reverse complementary to the forward or backward inner primers shown in FIG. Figure 62C shows a schematic representation of exemplary placement of guide RNAs relative to various regions of the nucleic acid sequences that correspond to or anneal to the LAMP primers. In this arrangement, the guide RNA hybridizes to sequences of the target nucleic acid that reside within the loop region between the B1 and B2 regions. The forward inner primer, backward inner primer, forward outer primer, and backward outer primer sequences do not contain PAM or PFS and are not reverse complementary to PAM or PFS. Figure 62D shows a schematic representation of exemplary placement of guide RNAs relative to various regions of the nucleic acid sequences that correspond to or anneal to LAMP primers. In this arrangement, the guide RNA hybridizes to sequences of the target nucleic acid that reside within the loop region between the F2c and F1c regions. The primer sequence does not contain PAM or PFS and is not reverse complementary to either PAM or PFS.
(FIG. 63) Nucleic acid sequences that correspond to or anneal to LAMP primer or guide RNA sequences, or that contain a protospacer flanking motif (PAM) or protospacer flanking site (PFS), and LAMP in combination for amplification and detection. Schematic representations of exemplary configurations of various regions of the target nucleic acid sequence for DETECTR and DETECTR, respectively. On the right, the schematic also shows the corresponding fluorescence data using LAMP amplification and guide RNA sequences to detect the presence of the target nucleic acid sequence, where the fluorescence signal is the output of the DETECTR reaction and the amount of the target nucleic acid. present. Figure 63A shows a schematic representation of the placement of various regions of the nucleic acid sequence that correspond to or anneal to the LAMP primers and the positions of the three guide RNAs (gRNA1, gRNA2 and gRNA3) relative to the LAMP primers (left). gRNA1 overlaps the B2c region and is therefore reverse complementary to the B2 region. gRNA2 overlaps the B1 region and is therefore reverse complementary to the B1c region. gRNA3 partially overlaps the B3 region and partially overlaps the B2 region, and is therefore partially reverse complementary to the B3c region and partially reverse complementary to the B2c region. Complementary regions (B1, B2c, B3c, F1, F2c and F3c) are not shown but correspond to the regions shown in FIG. On the right is a graph of fluorescence from DETECTR reactions in the presence of 10,000 genomic copies of target nucleic acid or 0 genomic copies of target nucleic acid. Figure 63B shows a schematic representation of the placement of various regions of the nucleic acid sequences that correspond to or anneal to the LAMP primers and the positions of the three guide RNAs (gRNA1, gRNA2 and gRNA3) relative to the LAMP primers (left). gRNA1 overlaps the B1c region and is therefore reverse complementary to the B1 region. gRNA2 overlaps the LF region and is therefore reverse complementary to the LFc region. gRNA3 partially overlaps the B2 region and partially overlaps the LBc region, and is therefore partially reverse complementary to the B2c region and partially reverse complementary to the LB region. On the right is a graph of fluorescence from DETECTR reactions in the presence of 10,000 genomic copies of target nucleic acid or 0 genomic copies of target nucleic acid. Figure 63C shows a schematic representation of the placement of various regions of the nucleic acid sequence that correspond to or anneal to the LAMP primers and the positions of the three guide RNAs (gRNA1, gRNA2 and gRNA3) relative to the LAMP primers (left). gRNA1 overlaps the B1c region and is therefore reverse complementary to the B1 region. gRNA2 partially overlaps the LF region and partially overlaps the F2c region, and is thus partially reverse complementary to the LFc region and partially reverse complementary to the F2 region. gRNA3 overlaps the B2 region and is therefore reverse complementary to the B2c region. On the right is a graph of fluorescence from DETECTR reactions in the presence of 10,000 genomic copies of target nucleic acid or 0 genomic copies of target nucleic acid.
(FIG. 64A) Nucleic acid sequences that correspond to or anneal to LAMP primer or guide RNA sequences, or contain protospacer flanking motifs (PAMs) or protospacer flanking sites (PFS), and target nucleic acids for LAMP and DETECTR assays shown in FIG. 63A. The arrangement of the various regions of the sequence and a detailed breakdown of the sequences are shown. Figure 64A discloses SEQ ID NO:393.
(FIG. 64B) Nucleic acid sequences that correspond to or anneal to LAMP primer or guide RNA sequences, or contain protospacer flanking motifs (PAMs) or protospacer flanking sites (PFS), and target nucleic acids for LAMP and DETECTR assays shown in FIG. 63B. The arrangement of the various regions of the sequence and a detailed breakdown of the sequences are shown. Figure 64B discloses SEQ ID NO:393.

(FIG. 64C) Nucleic acid sequences that correspond to or anneal to LAMP primer or guide RNA sequences, or contain protospacer flanking motifs (PAMs) or protospacer flanking sites (PFS), and target nucleic acids for LAMP and DETECTR assays shown in FIG. 63C. The arrangement of the various regions of the sequence and a detailed breakdown of the sequences are shown. Figure 64C discloses SEQ ID NO:393.
FIG. 65. Time to result of reverse transcription LAMP (RT-LAMP) reactions detected using DNA binding dyes.
(FIG. 66) Fluorescence signal from DETECTR reaction after 5 min incubation with product from RT-LAMP reaction. LAMP primer sets #1-6 in Figure 65 were designed for use with guide RNA #2 (SEQ ID NO:250) and LAMP primer sets #7-10 were designed for use with guide RNA #1 (SEQ ID NO:249). ) was designed to be used with
(FIG. 67) Influenza A using SYTO 9 (DNA binding dye) after RT-LAMP amplification with LAMP primer sets 1, 2, 4, 5, 6, 7, 8, 9, 10 or negative control Detection of sequences from viruses (IAV) is shown.
(FIG. 68) Shows time to amplification of influenza B virus (IBV) target sequences after RT-LAMP amplification. Amplification was detected using SYTO 9 in the presence of increasing concentrations of target sequence (0, 100, 1000, 10,000 or 100,000 genome copies of target sequence per reaction).
(FIG. 69) Shows time to amplification of IAV target sequences after LAMP amplification with different primer sets.
(FIG. 70) Influenza A virus (IAV)-derived using DETECTR after RT-LAMP amplification with LAMP primer sets 1, 2, 4, 5, 6, 7, 8, 9, 10, or negative control Detection of target nucleic acid sequences is shown. Ten reactions were performed for each primer set. The DETECTR signal was measured as a function of the amount of target sequence present in the reaction.
FIG. 71 shows a scheme for designing primers for LAMP amplification of a target nucleic acid sequence and detection of single nucleotide polymorphisms (SNPs) within the target nucleic acid sequence. In an exemplary arrangement, the SNP of the target nucleic acid is located between the F1c and B1 regions.
(FIG. 72) LAMP primers, guide RNA sequences, protospacer flanking motifs (PAMs) or protospacer flanking sites (PFS), and SNPs for methods of LAMP amplification of target nucleic acids and detection of target nucleic acids using DETECTR. FIG. 2 shows a schematic representation of an exemplary arrangement of target nucleic acids comprising; Figure 72A shows a schematic representation of exemplary placement of guide RNAs relative to various regions of the nucleic acid sequences that correspond to or anneal to the LAMP primers. In this arrangement the PAM or PFS of the target nucleic acid is located between the F1c and B1 regions. The entire guide RNA sequence can be present between the F1c and B1c regions. SNPs are shown as being located within the sequence of the target nucleic acid that hybridizes to the guide RNA. Figure 72B shows a schematic representation of exemplary placement of guide RNA sequences relative to various regions of the nucleic acid sequences that correspond to or anneal to LAMP primers. In this arrangement, the PAM or PFS of the target nucleic acid is positioned between the F1c and B1 regions, and the target nucleic acid comprises a sequence between the F1c and B1 regions that is reverse complementary to at least 60% of the guide nucleic acid. include. In this example, the guide RNA is not reverse complementary to the forward inner primer or the backward inner primer. SNPs are shown as being located within the sequence of the target nucleic acid that hybridizes to the guide RNA. Figure 72C shows a schematic representation of exemplary placement of guide RNA sequences relative to various regions of the nucleic acid sequences that correspond to or anneal to LAMP primers. In this arrangement, the PAM or PFS of the target nucleic acid is located between the F1c and B1 regions, and the entire guide RNA sequence may reside between the F1c and B1 regions. SNPs are shown as being located within the sequence of the target nucleic acid that hybridizes to the guide RNA.
(FIG. 73) Shows an exemplary sequence (SEQ ID NO:394) of a nucleic acid containing two PAM sites and a HERC2 SNP.
FIG. 74 shows the results of a DETECTR reaction to detect the HERC2 SNP at position 9 for the first PAM site or position 14 for the second PAM site after LAMP amplification. Fluorescent signals indicating detection of target sequences were measured over time in the presence of target sequences containing either the G or A allele in HERC2. Target sequences were detected using guide RNA (crRNA only) to detect either the A or G allele.
FIG. 75 shows a heat map of fluorescence from DETECTR reactions after LAMP amplification of target nucleic acid sequences. DETECTR reactions use guide RNA (crRNA only) specific for the A allele (SEQ ID NO:255, "R570 A SNP") or the G SNP allele (SEQ ID NO:256, "R571 G SNP") to discriminate between the two HERC2 alleles. Positive detection is indicated by high fluorescence values in the DETECTR reaction.
(FIG. 76) Combined LAMP amplification of target nucleic acid by LAMP and detection of target nucleic acid by DETECTR. Detection was done visually on the DETECTR by illuminating the sample with a red LED. Each reaction contained a target nucleic acid sequence containing a SNP allele for either the blue-eyed phenotype (“blue-eyed”) or the brown-eyed phenotype (“brown-eyed”). Samples "Brown*" and "Blue*" were the A and G allele positive controls, respectively. Guide RNA for either the brown-eyed (“Br”) or blue-eyed (“Bl”) phenotype was used for each LAMP DETECTR reaction.
(FIG. 77) Schematic showing steps for preparing and detecting the presence or absence of SARS-CoV-2 (“2019-nCoV”) in a sample using reverse transcription loop-mediated isothermal amplification (RT-LAMP), Cas12 , schematically shows the process of preparing and detecting the presence or absence of SARS-CoV-2 (“2019-nCoV reaction”).
(FIG. 78) SARS-CoV amplified with different primer sets (“2019-nCoV-set1” to “2019-nCoV-set12”) and detected using gRNAs directed to the N gene of LbCas12a and SARS-CoV-2 -2 shows the DETECTR assay results of the N gene. A shorter time to result indicates a positive result. For all primer sets, the time to result decreased the more target sequence was included in the sample, indicating that the assay was sensitive to the target sequence.
FIG. 79 shows individual traces of the DETECTR response plotted in FIG. 78 for the 0 fM and 5 fM samples. In each plot, the 0 fM trace was not visible above baseline, indicating little to no non-specific detection.
(FIG. 80) A primer set (“2019-nCoV-E-set13” to “ 2019-nCoV-E-set20") or amplified using primer sets directed to the N gene of SARS-CoV-2 (2019-nCoV-N-set21" to "2019-nCoV-N-set24") Shows the time to obtain the results of the DETECTR reaction on the sample. The best performing primer set for specific detection of the SARS-CoV-2 E gene was SARS-CoV-2-E-set14.
(FIG. 81) gRNA (“R1763-CDC-N2-Wuhan”) amplified with primer set 1 (“2019-nCoV-set1”) and directed to the N gene of LbCas12a and SARS-CoV-2 or SARS-CoV shows the DETECTR assay results of the SARS-CoV-2 N gene detected using one of the gRNAs directed against the N gene of 1 (“R1766-CDC-N2-SARS”).
(Figure 82) Determining the limit of detection of SARS-CoV-2 in DETECTR reactions amplified using a primer set directed to the N gene of SARS-CoV-2 ("2019-nCoV-N-set1") shows the results of the DETECTR reaction for Samples containing either 15,000, 4,000, 1,000, 500, 200, 100, 50, 20, or 0 copies of the SARS-CoV-2 N gene target nucleic acid were detected. A gel of the N gene RNA is shown below.
(Figure 83) Amplification of RNase P using the POP7 sample primer set. Samples were amplified using LAMP. DETECTR reactions were performed using a gRNA directed against RNase P (“R779”) and a Cas12 mutant (SEQ ID NO:37). Samples contained either HeLa total RNA or HeLa genomic DNA.
(FIG. 84) Shows time to results for multiplexed DETECTR reactions. The sample may be SARS-CoV-2 in vitro transcribed N gene (“N-gene IVT”), SARS-CoV-2 in vitro transcribed E gene (“E-gene IVT”), HeLa total RNA, or contained either no target (“NTC”). Samples were primed using one or more primer sets directed to the SARS-CoV-2 N gene (“set1”), SARS-CoV-2 E gene (“set14”), or RNase (“RNaseP”) amplified.
(Figure 85) Different combinations of primer sets directed to either the SARS-CoV-2 N gene ("set1"), the SARS-CoV-2 E gene ("set14"), or RNase P ("RNaseP"). Time to result for multiplexed DETECTR reaction used. Samples containing the in vitro transcribed N gene of SARS-CoV-2 (left, "N-gene IVT") or the in vitro transcribed E gene of SARS-CoV-2 (right, "E-gene IVT") tested.
FIG. 86 shows time to results for multiplexed DETECTR reactions using best performing primer set combinations from FIGS.
(FIG. 87A) Schematic representation of the CDC-N2 target site sequence used to detect the N-2 gene of SARS-CoV-2. Figure 87A discloses SEQ ID NOs: 395-398, respectively, in the order listed.
(FIG. 87B) Schematic representation of the sequence of the region of the SARS-CoV-2 N gene (“N-Sarbeco”) target site. Figure 87B discloses SEQ ID NOs: 399-400 and 400-401, respectively, in the order listed.
(Figure 88) N gene of SARS-CoV-2 (“N-2019-nCoV”), N gene of SARS-CoV (“N-SARS-CoV”) or N gene of bat-SL-CoV45 (“N- bat-SL-CoV45”), SARS-CoV-2 N gene (“R1763”), SARS-CoV N gene (“R1766”) or Salvecocoronavirus N gene (“R1763”) R1767'') shows the results of a DETECTR assay to determine the sensitivity of gRNAs directed to either
(FIG. 89) Schematic representation of the sequence of the region of the SARS-CoV-2 E gene (“E-Sarbeco”) target site. Figure 89 discloses SEQ ID NOs: 402-403 and 403-404, respectively, in the order listed.
(Figure 90) E gene of SARS-CoV-2 ("E-2019-nCoV"), E gene of SARS-CoV ("E-SARS-CoV"), E gene of bat-SL-CoV45 ("E- Determine the susceptibility of two gRNAs directed against the coronavirus N gene for samples containing either the E gene of bat-SL-CoV45") or bat-SL-CoV21 ("E-bat-SL-CoV21") shows the results of the DETECTR assay for
(FIG. 91) Shows the results of a lateral flow DETECTR reaction to detect the presence or absence of SARS-CoV-2 N gene target RNA using a Cas12 mutant (SEQ ID NO:37). 1 shows a lateral flow test strip. Samples containing (“+”) or lacking (“−”) in vitro transcribed SARS-CoV-2 N gene RNA (“N-gene IVT”) were tested. The upper set of horizontal lines (labeled "test") indicated the results of the DETECTR reaction.
(FIG. 92) Schematic representation of target nucleic acid detection using a programmable nuclease. Briefly, a Cas protein with trans-associated cleavage activity is activated upon binding to a guide nucleic acid and a target sequence that is reverse complementary to a region of the guide nucleic acid. An activated programmable nuclease cleaves the reporter nucleic acid, thereby generating a detectable signal.
(FIG. 93) Schematic representation of detection of the presence or absence of a target nucleic acid in a sample. Selected nucleic acids in the sample are amplified using isothermal amplification. The amplified sample is contacted with a programmable nuclease, guide nucleic acid and reporter nucleic acid as shown in FIG. If the sample contains the target nucleic acid, a detectable signal is produced.
(FIG. 94) Shows the results of a DETECTR lateral flow reaction to detect the presence or absence of SARS-CoV-2 (“2019-nCoV”) RNA in a sample. Detection of RNase P is used as sample quality control. Samples were transcribed and amplified in vitro (left) and detected using Cas12 programmable nuclease (right). Samples containing (“+”) or lacking (“−”) in vitro transcribed SARS-CoV-2 RNA (“2019-nCoV IVT”) were treated with Cas12 programmable nuclease and gRNA directed against SARS-CoV-2. was assayed for 0 or 5 minutes using Reactions were sensitive to samples containing SARS-CoV-2.
(FIG. 95) Shows the results of a DETECTR reaction using the LbCasl2a programmable nuclease (SEQ ID NO:27) to determine the presence or absence of SARS-CoV-2 in patient samples.
(FIG. 96) Shows the results of a lateral flow DETECTR reaction to detect the presence or absence of SARS-CoV-2 in patient samples. Samples were detected with either gRNAs directed against SARS-CoV-2 or gRNAs directed against RNase P.
(FIG. 97) Technical specifications and assay conditions for detection of coronavirus using reverse transcription and loop-mediated isothermal amplification (RT-LAMP) and Cas12 detection.
FIG. 98 shows the results of a DETECTR assay evaluating multiple gRNAs for detection of SARS-CoV-2 using LbCasl2a. The target nucleic acid sequence was selected from the SARS-CoV-2 E gene (“2019-nCoV-E-set13” to “2019-nCoV-E-set20” or the SARS-CoV-2 N gene (“2019-nCoV-N-set21” "2019-nCoV-N-set24" from ) was amplified using a primer set for amplification.
(FIG. 99) A DETECTR assay evaluating multiple gRNAs for utility in distinguishing between three different strains of coronavirus, SARS-CoV-2 (“COVID-2019”), SARS-CoV, or bat-SL-CoV45. shows the results of N of either SARS-CoV-2 (“N-2019-nCoV”), SARS-CoV (“N-SARS-CoV”), or bat-SL-CoV45 (“N-bat-SL-CoV45”) Samples containing gene amplicons were tested.
(FIG. 100) DETECTR assay evaluating multiple gRNAs for utility in distinguishing between three different strains of coronavirus, SARS-CoV-2 (“COVID-2019”), SARS-CoV, or bat-SL-CoV45. shows the results of E for either SARS-CoV-2 (“N-2019-nCoV”), SARS-CoV (“N-SARS-CoV”), or bat-SL-CoV45 (“N-bat-SL-CoV45”) Samples containing gene amplicons were tested.
(FIG. 101) Shows the results of a DETECTR assay evaluating LAMP primer sets for their utility in multiplexed amplification of SARS-CoV-2 targets. Using one or more primer sets directed to the SARS-CoV-2 N gene (“set1”) or SARS-CoV-2 E gene (“set14”) or RNase P (“RNaseP”) amplified.
(FIG. 102) Shows the results of a DETECTR assay assessing the sensitivity of RT-LAMP amplification reactions to common sample buffers. Various buffer dilutions (left to right: 1×, 0.5×, 0.25×, 0.125×, or no buffer) in Universal Transport Medium (UTM, top) or DNA/RNA Shield buffer (bottom) The response was measured at .
(FIG. 103) Shows the results of a DETECTR assay to determine the limit of detection (LoD) of the DETECTR assay for SARS-CoV-2, the virus caused by COVID-19 infection.
(FIG. 104) Gene expression of gRNA directed to the N gene of SARS-CoV-2 (“R1763-N-gene”) in a 2-plex multiplexed RT-LAMP reaction using the LbCas12a programmable nuclease (SEQ ID NO:27). Results of a DETECTR assay to assess target specificity are shown.
(FIG. 105) SARS-CoV-2 N gene (“R1763-N-gene”) or SARS-CoV- 2 shows the results of a DETECTR assay evaluating the target specificity of gRNAs directed to the E gene of 2 (“R1765-E-gene”).
(FIG. 106) Design of a detector nucleic acid compatible with the PCRD lateral flow device. Exemplary matched detector nucleic acids, rep072, rep076, and rep100 are provided (left). These detector nucleic acids can be used in a PCRD lateral flow device (right) to detect the presence or absence of target nucleic acids. The upper right schematic shows an exemplary band composition produced when contacted with a sample containing no target nucleic acid. The bottom right schematic shows an exemplary band composition produced upon contact with a sample containing a target nucleic acid. Figure 106 discloses SEQ ID NOs: 9, 372, and 372, respectively, in the order listed.
(FIG. 107) FIG. 107A shows a genomic map showing the location of the E (envelope) gene and N (nucleoprotein) gene regions within the genome of coronaviruses. The corresponding or annealing regions of the primers and probes for the E and N gene regions are shown below the respective gene regions. The RT-LAMP primer is indicated by a black rectangle, and the binding position of the FIP primer (grey) with half of F1c and B1c is represented by a striped rectangle with dashed borders. The regions amplified in tests used by the World Health Organization (WHO) and the Centers for Disease Control (CDC) are called "WHO E amplicon" and "CDC N2 amplicon" respectively. Figure 107B shows the use of the LbCas12a programmable nuclease (SEQ ID NO:27) to target the N or E gene of various coronavirus strains (SARS-CoV-2, SARS-CoV, or bat-SL-CoVZC45). Results of DETECTR assays assessing the specificity or broad detection utility of gRNAs are shown. The N-gene gRNA (left, 'N-gene') used in the assay was specific for SARS-CoV-2, whereas the E-gene gRNA was able to detect three SARS-like coronaviruses (right). , 'E gene'). Separate N-gene gRNAs targeting SARS-CoV and bat coronavirus failed to detect SARS-CoV-2 (middle, 'N-gene-associated species variant'). Figure 107C shows an exemplary experimental setup used in the coronavirus DETECTR assay. In addition to appropriate biosafety protection equipment, the equipment used includes a sample collection device, microcentrifuge tubes, heat blocks set at 37°C and 62°C, pipettes and tips, and lateral flow strips. FIG. 107D shows an exemplary workflow of a DETECTR assay for detection of coronavirus in a subject. Conventional RNA extraction or sample matrix can be used as input to DETECTR (LAMP pre-amplification and Cas12-based detection of NE genes, EN genes and RNase P), which is visualized by fluorescence readers or lateral flow strips. be. Figure 107E shows a lateral flow test strip (left) showing a positive test result for the SARS-CoV-2 N gene (left, top) and a negative test result for the SARS-CoV-2 N gene (left, bottom). show. Table (right) shows possible test indicators and potential test indicators for a lateral flow strip-based coronavirus diagnostic assay testing for the presence of RNase P (positive control), the SARS-CoV-2 N gene, and the absence of the coronavirus E gene. Show the relevant results.
(FIG. 108) FIG. 108A shows cleavage of a FAM- and biotin-labeled detector nucleic acid by Cas12 programmable nuclease in the presence of target nucleic acid (top). A schematic of a lateral flow test strip (bottom) shows markings that indicate either the presence (“positive”) or absence (“negative”) of target nucleic acid in the test sample. The intact FAM-biotinylated reporter molecule flows into the control capture line. Upon recognition of a matching target, the Cas-gRNA complex cleaves the reporter molecule, which flows into the target capture line. FIG. 108B shows the results of a DETECTR assay using LbCasl2a to determine the effect of reaction time on samples containing either 0 fM SARS-CoV-2 RNA or 5 fM SARS-CoV-2 RNA. For the SARS-CoV-2 N gene, the fluorescence signal of the LbCas12a detection assay with the RT-LAMP amplicon saturated within 10 minutes. RT-LAMP amplicons were generated from 2 μL of 5 fM or 0 fM SARS-CoV-2 N-gene IVT RNA by amplification for 20 minutes at 62°C. Figure 108C shows a lateral flow test strip assaying samples equivalent to those assayed by the DETECTR of Figure 108B. For samples containing either 0 fM SARS-CoV-2 RNA (“-”) or 5 fM SARS-CoV-2 RNA (“+”), bands corresponding to control (C) or test (T) as a function of reaction time. LbCas12a on the same RT-LAMP amplicon produced a visible signal throughout the lateral flow assay within 5 minutes. FIG. 108D shows the results of DETECTR assays using LbCasl2a (middle) or CDC protocol (left) to determine the limit of detection for SARS-CoV-2. Signal is shown as a function of viral genome copy number per reaction. Representative lateral flow results of the assay are shown for 0 copies/μL and 10 copies/μL (right). FIG. 108E shows DETECTR data for patient samples. Six patients with COVID-19 infection (n=11, repeat 5) and influenza or 1 of 4 seasonal coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43) Clinical samples from 12 patients (n = 12) with HIV were analyzed using the SARS-CoV-2 DETECTR (shaded boxes). Signal intensities from lateral flow strips were quantified using ImageJ and normalized to the maximum value within the N gene, E gene or RNase P set using a positive threshold of 5 standard deviations above background. The final decision for SARS-CoV-2 testing was based on the interpretation matrix of Figure 107E. FluA stands for Influenza A and FluB for Influenza B. HCoV stands for human coronavirus. Figure 108F shows a COVID-19 patient (positive for SARS-CoV-2, "Patient 1"), a no-target control sample lacking the target nucleic acid ("NTC"), and a positive control sample containing the target nucleic acid ("PC") shows a lateral flow test strip testing for SARS-CoV-2. All three samples were tested for the presence of SARS-CoV-2 N gene, SARS-CoV-2 E gene, and RNase P. Figure 108G shows the performance characteristics of the SARS-CoV-2 DETECTR assay. Eighty-three clinical samples (41 positive, 42 negative for COVID-19) were evaluated using the fluorescence version of the SARS-CoV-2 DETECTR assay. One sample (COVID19-3) was excluded due to assay quality control failure. Positive and negative calls were based on the criteria described in Figure 32E. fM indicates femtomoles, NTC indicates no template control, PPA indicates positive predictive agreement, NPA indicates negative predictive agreement.
(FIG. 109) Table comparing SARS-CoV-2 DETECTR assay using RT-LAMP of the present disclosure with SARS-CoV-2 assay using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detection method. indicates The N gene target in the DETECTR RT-LAMP assay is the same as the N gene N2 amplicon detected in the qRT-PCR assay.
(FIG. 110A) Time to result of RT-LAMP amplification under different buffer conditions. Time to result was calculated as the time at which the fluorescence value was ⅓ of the experimental maximum. Reactions that failed amplification are reported as 20 minute values and labeled as "no amp". Time to results was determined for different starting concentrations of target control plasmid in either water, 10% phosphate buffered saline (PBS), or 10% universal transport medium (UTM). A shorter time to result indicates faster amplification.
(FIG. 110B) RT-LAMP assay to determine amplification efficiency of SARS-CoV-2 N gene, SARS-CoV-2 E gene and RNase P in either 5% UTM, 5% PBS or water. shows the results of Samples containing 0.5 fM in vitro transcribed N gene, 0.5 fM in vitro transcribed E gene, and 0.8 ng/μL HeLa total RNA (“N+E+total RNA”) or no target control (“NTC”) ) was tested.
(FIG. 110C) Direct RNA amplification from nasal swabs in PBS. Time to result was measured as a function of PBS concentration. Nasal swabs (“nasal swabs”) were spiked with either HeLa total RNA (left, “total RNA: 0.08 ng/uL”) or water (right, “total RNA: 0 ng/uL”). A sample without a nasal swab (“no swab”) was compared as a control.
(FIG. 111A) Raw fluorescence curves generated by LbCasl2a (SEQ ID NO:27) detection of the SARS-CoV-2 N gene (n=6). The curve showed saturation in less than 20 minutes.
(FIG. 111B) Detection limit of DETECTR assay for LbCas12a-detected SARS-CoV-2 N gene determined from the raw fluorescence trace shown in FIG. 111A. Fluorescence intensity was measured with decreasing concentrations (copies/mL) of the SARS-CoV-2 N gene.
(FIG. 111C) Shows the time to detection limit results of the DETECTR assay as determined from the raw fluorescence trace shown in FIG. 111A. A shorter time to result indicates faster detection.
FIG. 112A depicts the DETECTR assay using LbCas12a to determine the effect of reaction time on samples containing either 0 fM SARS-CoV-2 RNA or 5 fM SARS-CoV-2 RNA. Show the results. FIG. 112B shows a lateral flow test strip assaying samples equivalent to those assayed by the DETECTR of FIG. 112A. For samples containing either 0 fM SARS-CoV-2 RNA (“-”) or 5 fM SARS-CoV-2 RNA (“+”), bands corresponding to control (C) or test (T) as a function of reaction time.
(FIG. 113) Shows the results of a DETECTR assay to determine the cross-reactivity of gRNAs to different human coronavirus strains. Contains in vitro transcribed RNA of SARS-CoV-2 N gene, SARS-CoV N gene, bat-SL-CoVZC45 N gene, SARS-CoV-2 E gene, SARS-CoV E gene or bat-SL-CoVZC45 E gene Samples or clinical samples positive for CoV-HKU1, CoV-299E, CoV-OC43 or CoV-NL63 were tested. HeLa total RNA was tested as a positive control for RNase P and a sample lacking target nucleic acid ("NTC") was tested as a negative control.
FIG. 114A shows a sequence alignment of the target sites targeted by the N-gene gRNAs (SEQ ID NOs: 405-410, respectively, in the order listed) for three coronavirus strains. N-gene gRNA#1 is compatible with the CDC-N2 amplicon and N-gene gRNA#2 is compatible with the WHO N-Sarbeco amplicon. Figure 114B shows a sequence alignment of the target sites targeted by the E gene gRNAs (SEQ ID NOS: 411-416, respectively, in the order listed) for three coronavirus strains. Two E-gene gRNAs tested (E-gene gRNA#1 and E-gene gRNA#2) are compatible with the WHO E-Sarbeco amplicon.
(FIG. 115) FIGS. 115A-115C show DETECTR kinetic curves for COVID-19 infected patient samples. Ten nasal swab samples from five patients (COVID19-1 to COVID19-10) were tested for SARS-CoV-2 using two different genes, N2 and E, and a sample input control, RNase P did. Figure 115A shows that using standard amplification and detection conditions, 9 out of 10 patients produced strong fluorescence curves indicating the presence of the SARS-CoV-2 E gene (20 min. amplification, signal within 10 min). Figure 115B shows that the amplification time required to generate a strong fluorescence curve for the SARS-CoV-2 N gene was prolonged for 8 out of 10 patients (30 min amplification, 10 min signal within). Figure 115C shows that RNase P as a sample input control was positive in 17 of all 22 samples tested (20 min amplification, signal within 10 min).
(FIG. 116) DETECTR analysis of SARS-CoV-2 shows that up to 10 viral genomes are identified in approximately 30 minutes (20 minutes amplification, 10 minutes DETECTR). Duplicate LAMP reactions were amplified for 20 minutes prior to LbCas12a DETECTR analysis.
(FIG. 117) Raw fluorescence at 5 min for the LbCas12a DETECTR analysis provided in FIG. The limit of detection for the SARS-CoV-2 N gene was determined to be 10 viral genomes per reaction (n=6).
(FIG. 118) Lateral flow DETECTR results for 10 COVID-19 infected patient samples and 12 patient samples for other viral respiratory infections. Ten samples (COVID19-1 to COVID19-5) from 6 patients, including 1 nasopharyngeal swab (A) and 1 oropharyngeal swab (B), were analyzed for two different genes, N2 and E, and Sample input control, RNase P was used to test for SARS-CoV-2. Results were analyzed according to the guidelines provided in FIG.
(Figure 119) Shows the rules for the interpretation of SARS-CoV-2 DETECTR lateral flow results.
(FIG. 120A) Fluorescence DETECTR kinetic curves of 11 COVID-19 infected patient samples and 12 patient samples with other viral respiratory infections. Ten nasopharyngeal/oropharyngeal swab samples (COVID19-1 to COVID19-6) from 6 patients were tested for SARS-CoV using two different genes, N2 and E, and a sample input control, RNase P -2 tested. Figure 120A shows samples tested using standard amplification and detection conditions, with strong fluorescence curves showing the presence of the SARS-CoV-2 E gene in 10 out of 12 COVID-19 positive patient samples. resulted (20 min amplification, signal within 10 min). No E gene signal was detected in 12 other viral respiratory clinical samples.
(FIG. 120B) Fluorescence DETECTR kinetic curves for 11 COVID-19 infected patient samples and 12 patient samples with other viral respiratory infections. Ten nasopharyngeal/oropharyngeal swab samples (COVID19-1 to COVID19-6) from 6 patients were tested for SARS-CoV using two different genes, N2 and E, and a sample input control, RNase P -2 tested. FIG. 120B shows that for 10 of 12 COVID-19 positive patient samples tested for the presence of the SARS-CoV-2 N gene using time-extended amplification produce strong fluorescence curves. (amplification for 30 minutes, signal within 10 minutes). No N gene signal was detected in 12 other viral respiratory clinical samples.
(FIG. 120C) Fluorescence DETECTR kinetic curves of 11 COVID-19 infected patient samples and 12 patient samples with other viral respiratory infections. Ten nasopharyngeal/oropharyngeal swab samples (COVID19-1 to COVID19-6) from 6 patients were tested for SARS-CoV using two different genes, N2 and E, and a sample input control, RNase P -2 tested. Figure 120C shows a graph corresponding to RNase P, the sample input control.
FIG. 121 shows a heatmap of SARS-CoV-2 DETECTR assay results for clinical samples with study interpretations indicated. 24 clinical samples (COVID-19 positive, 12) were quantified for SARS-CoV-2 DETECTR by the ImageJ gel analyzer tool. Shows 98.6% (71/72 strips) agreement with the fluorescence version results (bottom). Both assays were run with 30 minutes of amplification and Cas12 response signal was obtained at 10 minutes. Putative positives are indicated by an orange (+) (bottom, row 4).
FIG. 122 shows a heatmap of SARS-CoV-2 DETECTR assay results for clinical samples with study interpretations indicated. The top plot shows the results of the fluorescent SARS-CoV-2 DETECTR assay on an additional 30 COVID-19 positive clinical samples (27 positive, 1 probable positive, 2 negative). Putative positives are indicated by an orange (+) (top, column 9). The bottom plot shows the results of the fluorescent SARS-CoV-2 DETECTR assay on an additional 30 COVID-19 negative clinical samples (0 positive, 30 negative).
FIG. 123 shows time to results for RT-LAMP amplification of RNase P POP7 using different primer sets. Time to results was determined for samples amplified with primer sets 1-10. Primer set 1 corresponds to SEQ ID NO:360-SEQ ID NO:365 and primer set 9 corresponds to SEQ ID NO:366-SEQ ID NO:371.
(FIG. 124) Raw fluorescence over time of DETECTR reaction performed on RNase P POP7 amplified using RT-LAMP with primer set 1 or primer set 9 and detected with R779, R780 or R1965 gRNA. show. The DETECTR reaction was performed at 37°C for 90 minutes. Amplicons generated by Set 1 primers were detected with no background (dotted line) by R779.
Figure 125A shows time to results for RNase P POP7 detection in samples containing 10-fold dilutions of total RNA amplified using RT-LAMP with primer set 1 or primer set 9. . Amplification was performed at 60°C for 30 minutes. Figure 125B shows the DETECTR reaction of the RNase P POP7 amplicon shown in Figure 125A and detected using gRNA 779 (SEQ ID NO:330) or gRNA 1965 (SEQ ID NO:331). Samples amplified using primer set 1 were detected with gRNA 779, and samples amplified using primer set 9 were detected with gRNA 1965. The DETECTR reaction was performed at 37°C for 90 minutes.
(FIG. 126) FIGS. 126A and 126B show photographs of cartridges designed for use in the DETECTR assay.
(FIG. 127) FIGS. 127A and 127B are schematic diagrams of the cartridge shown in the photograph of FIG. 126A.
(FIG. 128A) Schematic representation of a cartridge designed for use in the DETECTR assay. FIG. 128A shows a cartridge with circular reagent storage wells and a high resistance tortuous path in the z direction.
(FIG. 128B) Schematic representation of a cartridge designed for use in the DETECTR assay. FIG. 128B shows a cartridge with elongated reagent storage wells and high resistance tortuous paths in the z direction.
(FIG. 128C) Schematic representation of a cartridge designed for use in the DETECTR assay. FIG. 128C shows a cartridge with circular reagent storage wells and high resistance tortuous paths in the xy directions.
(FIG. 128D) Schematic representation of a cartridge designed for use in the DETECTR assay. FIG. 128D shows a cartridge with elongated reagent storage wells and high resistance tortuous paths in the xy directions.
(FIG. 129) FIGS. 129A-129D show schematics of cartridges designed for use in DETECTR assays. FIG. 129A shows a cartridge with a meander-resistant channel for sample metering that meanders on a different plane or layer than the sample metering channel. FIG. 129B shows a cartridge with a meander-resistant channel for sample metering that meanders on the same plane or layer as the sample metering channel. FIG. 129C shows a cartridge containing a resistive path for arduous path sample metering at right angles and a DETECTR sample metering inlet on a different plane or layer than the sample metering channel. FIG. 129D shows a cartridge containing a right-angled hard-path resistant path for sample metering and a DETECTR sample metering inlet on the same plane or layer as the sample metering channel.
(Figure 130) Figure 130A shows features of a cartridge designed for use in the DETECTR assay. FIG. 130B shows a manufacturing scheme (left and middle) for manufacturing a cartridge of the present disclosure and a readout device (right) for detecting the sample in the cartridge.
(FIG. 131) FIG. 131A shows a schematic view of a cartridge manifold for heating regions of the cartridge of the present disclosure. The cartridge manifold has an integrated heating zone with an integrated air supply connection and an integrated O-ring groove for the air supply interface. The cartridge manifold thermally isolates the amplification temperature zone from the detection temperature zone and includes isolation zones for maintaining appropriate temperatures in the amplification and detection chambers of the cartridge. FIG. 131B shows two manufacturing methods for manufacturing the cartridges described herein. The first manufacturing method (left) uses a two-dimensional (2D) stack of multiple layers to manufacture the cartridge. In the second manufacturing method (right), parts containing integrated complex features are injection molded and sealed by lamination. FIG. 131C shows a schematic of a cartridge with a luer slip adapter for coupling the cartridge to a syringe. The adapter can form an interference fit seal with a slip luer tip. The adapter is configured to work with any of the cartridges disclosed herein.
(FIG. 132) FIGS. 132A and 132B show schematics of an integrated flow cell for use with a microfluidic cartridge. An integrated flow cell contains three areas: a lysis area, an amplification area, and a detection area. The lysis area is long enough to accommodate a microfluidic chip shop sample lysis flow cell. Lysis flow cells may be combined with amplification and detection chambers on the cartridges disclosed herein.
(FIG. 133) Detail of an input channel on a cartridge of the present disclosure.
(FIG. 134) Shows a workflow for performing a DETECTR assay using a microfluidic cartridge of the present disclosure. A cartridge (“chip”) is filled with sample and reaction solution. Heat the amplification chamber (“LAMP chamber”) to 60 °C and incubate the sample in the amplification chamber for 30 min. The amplified sample (“LAMP amplicon”) is pumped into the DETECTR reaction chamber and the DETECTR reagent is pumped into the DETECTR reaction chamber. Heat the DETECTR reaction chamber to 37 °C and incubate the sample for 30 min. Fluorescence in the DETECTR reaction chamber is measured in real time to generate quantitative results.
FIG. 135 shows a schematic diagram of the structure of the system electronics of a cartridge manifold compatible with the cartridges disclosed herein. The electronics are configured to heat a first zone of the cartridge to 37°C and a second zone of the cartridge to 60°C.
(FIG. 136) FIGS. 136A and 136B show schematic diagrams of cartridge manifolds for heating and sensing cartridges of the present disclosure. The manifold is configured to receive the cartridge, facilitate the DETECTR reaction, and read the fluorescence resulting from the DETECTR reaction.
(FIG. 137A) An example of a fluorescent sample in a cartridge, illuminated with a cartridge manifold. Positive control wells contain reagents and amplified samples after a 30 minute amplification step at 60°C and a 30 minute detection step at 37°C. Empty wells serve as false negative samples.
(FIG. 137B) Cartridge manifold for heating and sensing cartridges of the present disclosure.
(FIG. 137C) Cartridge manifold for heating and sensing cartridges of the present disclosure.
(FIG. 138) FIGS. 138A and 138B show fluorescence generated within the detection chamber of a microfluidic cartridge facilitated by manifolds of the present disclosure.
(Fig. 139) Figs. 139A and 139B show a thermal test schematic for an amplification chamber heated to 60°C (Fig. 139A) or a DETECTR chamber heated to 37°C (Fig. 139B).
(Fig. 140) Figs. 140A and 140B show a thermal test schematic for an amplification chamber heated to 60°C (Fig. 140A) or a DETECTR chamber heated to 37°C (Fig. 140B).
(FIG. 141) FIG. 141A shows DETECTR results run on a plate reader with a gain of 100 using the LAMP product from the microfluidic cartridge as input. Samples were run in duplicate with a single non-template control (NTC). Figure 141B shows three LAMP products performed on a plate reader using samples from the microfluidic chip. LAMP reactions are numbered in the order in which the chip was run (LAMP_1 being run first, etc.). The donor is homozygous for SNP A, so crRNA 570 appears first. ATTO 488 was used as fluorescence standard.
(FIG. 142) FIG. 142A shows an image of a filled microfluidic chip. Figure 142B shows the results of the DETECTR reaction measured with a plate reader after 30 minutes of LAMP amplification.
(Figure 143) Figures 143A, 143B, 143C and 143D show the results of the coronavirus DETECTR reaction. Two reaction chambers with 10 copies input to LAMP resulted in a rapid increase in DETECTR signal. All NTCs were negative. When 10 copies were input to the LAMP, the DETECTR signal gradually increased over the course of the reaction, as shown in the photodiode measurements in Figure 143C below. The negative control in Figure 143D showed no contamination.
(Figure 144) Figures 144A, 144B, 144C and 144D show the results of repeated coronavirus DETECTR reactions.
(FIG. 145) FIGS. 145A and 145B show photodiode measurements of influenza B DETECTR reactions in microfluidic cartridges.
(FIG. 146) FIGS. 146A, 146B and 146C show photodiode measurements of influenza B DETECTR responses in microfluidic cartridges.
(FIG. 147) Fluorescence results from a series of DETECTR reagents stored in glass capillaries for 7 months.
(FIG. 148) Provides spin-through column designs and methods of using spin-through columns for serial amplification and DETECTR reactions.
(FIG. 149) Provides structures of three reagents used to construct electrochemically detectable nucleic acids: (A) ferrocene-labeled thymidine, (B) 6-carboxyfluorescein, and (C) biotin-labeled phosphate. do.
(FIG. 150) Provides an injection molded cartridge design that includes a sample input chamber and multiple chambers in which a portion of the sample can be subjected to amplification and detector reactions.
(FIG. 151) Provides a device design including a detector diode array and heating panel that can use the injection molded cartridge shown in FIG.
(FIG. 152) Fluorescence data from a series of DETECTR reactions performed on samples subjected to different dual lysis amplification buffers.
(FIG. 153) Fluorescence data from a series of DETECTR reactions performed on samples subjected to different dual lysis amplification buffers.
(FIG. 154) Panel (a) provides an injection molded cartridge design for performing multiple amplification and DETECTR reactions on a sample. Panel (b) provides the design of an instrument configured to use an injection-molded cartridge and measure the fluorescence from the DETECTR reaction performed in the cartridge.
(FIG. 155) A method is provided for using injection molded cartridges and the apparatus shown in FIG. 154 to perform parallel amplification and DETECTR reactions on samples.
FIG. 156 shows the injection molded cartridge and diode array and dye-filled reaction compartments from the apparatus of FIG.
(FIG. 157) Shows a possible design of an injection molded cartridge containing one sample chamber connected to five amplification chambers and two detection chambers connected to each amplification chamber. Therefore, the instrument can perform 10 parallel DETECTR reactions on a single sample.
(FIG. 158) Shows a possible design of an injection molded cartridge containing one sample chamber connected to four amplification chambers and two detection chambers connected to each amplification chamber. The injection molded cartridge contains a series of valves and pumps or ports for pumping a pump manifold that controls flow throughout the cartridge.
(Fig. 159) A possible design of an injection molded cartridge containing one sample chamber connected to four amplification chambers, two detection chambers connected to each amplification chamber, and a reagent chamber in contact with the sample chamber. show.
(FIG. 160) Provides a top view of an injection molded cartridge design with reagent chambers within flow paths leading to amplification and detection chambers.
(FIG. 161) Part of an injection molded cartridge design with a sample chamber that can be connected to multiple reagent and amplification chambers by a single rotary valve.
(FIG. 162) Part of an injection molded cartridge design with a slide valve connecting multiple compartments. Panels A-C show various positions the slide valve can choose from.
(FIG. 163) Panel A shows a possible design of an injection molded cartridge with a casing. Panel B provides a physical model of the design shown in panel A.
(FIG. 164) Panel A provides a bottom view of the injection molded cartridge design with casing. Panel B provides a view of the top of the injection molded cartridge.
(FIG. 165) Provides multiple views of an injection molded cartridge with a slide valve.
(FIG. 166) Provides two views of a portion of an injection molded cartridge with multiple reagent wells leading to a transparent reaction chamber.
(FIG. 167) Panels AB provide a top view of the injection molded cartridge design. Panel C shows a photograph of the physical model of the injection molded cartridge.
(FIG. 168) Shows a photograph of an injection molded cartridge housed in a device containing a diode array.
FIG. 169 shows a graphical user interface for controlling a device containing an injection molded cartridge and a diode array for detection.
FIG. 170 shows the results of a series of fluorescence experiments using an 8-diode detector array, an 8-chamber injection molded cartridge, and dyes.
(FIG. 171) Fluorescence results from a series of HERC2 targeting DETECTR reactions and buffer controls measured using an 8-diode detector array.
(FIG. 172) Shows an injection molded cartridge inserted into the device with eight chambers containing DETECTR reactions.
(FIG. 173) Shows the results of amplification of SeraCare target nucleic acids using LAMP under different lysis conditions. Samples were amplified in low pH buffer containing either buffer (upper plot) or virus lysis buffer (“VLB”, lower plot). Buffers contained no reducing agent (“control”, columns 1 and 4), reducing agent B (columns 2 and 5), or reducing agent A (columns 3 and 6). Samples were incubated for 5 min at either room temperature (left plot) or 95 °C (right plot). Samples contain either no target (“NTC”) or 2.5, 25, or 250 copies per reaction. Assays were performed in triplicate using 5 μL of sample in 25 μL reactions.
(FIG. 174) Results of amplification of SeraCare standard target nucleic acid using LAMP under different lysis conditions. Samples were amplified in low pH buffer containing either buffer (left plot) or virus lysis buffer (“VLB”, right plot). Buffers contained no reducing agent (“control”), reducing agent B, or reducing agent A. Samples were incubated for 5 min at either room temperature (upper plot) or 95 °C (lower plot). Samples contain either no target (“NTC”) or 1.5, 2.5, 15, 25, 150, or 250 copies per reaction. Assays were performed in triplicate using 3 μL of sample in a 15 μL reaction or 5 μL of sample in a 25 μL reaction.
(Figure 175) in the presence of six different virus lysis buffers ("VLB", "VLB-D", "VLB-T", "buffer", "buffer A", "buffer B") , shows amplification of the SARS-CoV-2 N gene (“N”) and RNase P sample input control nucleic acid (“RP”). Buffer-A contains a buffer containing reducing agent A and buffer-B contains a buffer containing reducing agent B. Shaded squares indicate amplification rates, darker shading indicating faster amplification. Amplification was performed at 95°C (“95 C”) or room temperature for high, medium or low titer COVID-19 positive patient samples (“16.9”, “30.5” and “33.6” respectively). (“RT”). Samples were measured in duplicate.
FIG. 176 shows square wave voltammetry results of DETECTR reactions performed with electroactive reporter nucleic acids. Results were collected immediately after initiation of the DETECTR reaction (0 minutes) and 33 minutes later.
FIG. 177 shows cyclic voltammetry results of DETECTR reactions performed with electroactive reporter nucleic acids. Results were collected immediately after initiation of the DETECTR reaction (0 minutes) and 26 minutes later.

(Table 4) Cas13 protein sequence

(Table 6) Exemplary LAMP Primers

Cas13M26 (LbuCas13a (SEQ ID NO:131)) performs optimally in DETECTR reactions in buffers with reduced amounts of tRNA without altering reaction stability. Reducing the amount of tRNA in the reaction or eliminating it entirely increases the efficiency of the Cas13a detection assay without dramatically altering the stability of the reaction in the absence of activator. Buffers in which Cas13a is active in DETECTR assays for detecting nucleic acids from respiratory viruses (eg, influenza virus) lack or contain small amounts of urea and SDS. In addition, Cas13a is inhibited by respiratory viruses (e.g., It exhibits activity in the DETECTR assay for detecting nucleic acids from influenza virus). Cas13a also has a 'U5' reporter (/5-6FAM/rUrUrUrUrU/3IABkFQ/ (SEQ ID NO: 1) ), a 'UU' reporter (/56-FAM/TArUrUGC/3IABkFQ/ ( SEQ ID NO: 1)) when measured by fluorescence. ID NO: 381) ), and the reporter 'TYE665U5' (/5-TYE665/rUrUrUrUrU/3IABkRQ/ (SEQ ID NO: 1) ), which has the same nucleotide sequence as the 'U5' reporter but a different fluorophore and quencher. It exhibits activity for many reporters, including Optimal buffer composition and pH for Cas13a DETECTR assays include buffers having a pH of about 7.5 and buffered imidazole, phosphate, tricine and SPG.

Lateral flow strips for readout of LbuCas13a. Test on lateral flow strips for readout of LbuCas13a. FAM-U5-biotin (SEQ ID NO: 1) (rep71 reporter) is tested using TwistDx lateral flow strips. Assays are performed at room temperature at various target concentrations. Conjugation reactions contain final concentrations of 40 nM crRNA per reaction, 40 nM final protein per reaction, and 500 nM reporter per reaction. The conjugation reaction is incubated at 37°C for 30 minutes. Dilutions of 10 nM, 1 nM, 0.1 nM, 0.01 nM of target and no target are added to the reactions. Add 30 uL of the conjugation reaction to the target and incubate for 15 minutes at room temperature. Place the reaction on ice and pipet 10 uL of the reaction directly into the lateral flow sample area. 50 uL of Milenia GenLine Dipstick Assay Buffer was added and the strips were photographed.

（SEQ ID NO：372）である。
Conjugation of 3' amino reporter to NHS beads using kit. The NHS FlexiBind magnetic bead kit was used to conjugate a 3' amino-modified lateral flow reporter that allows for the intended use of the lateral flow device (Milenia Hybrid), where the ligand is first detected and the control line is flowed. Act as a control. The array of reporters used is

(SEQ ID NO: 372) .

(Table 12) Reporter sequences

(Table 14) LAMP primers for RT-LAMP amplification and detection

For trans-cleavage of LbCas12a (SEQ ID NO:27), 50 nM LbCas12a (available from NEB) was pre-incubated with 62.5 nM gRNA in 1×NE buffer 2.1 at 37° C. for 30 minutes. After formation of the RNA-protein complex, a lateral flow cleavage reporter (/56-FAM/TTATTATT/3Bio/ (SEQ ID NO:9) , IDT) was added to the reaction at a final concentration of 500 nM. RNA-protein complexes were used immediately or stored at 4°C for up to 24 hours before use.

A patient-optimized fluorescence-based LbCas12a transcleavage assay was performed as described above with the following modifications; 100 nM of a fluorescent reporter molecule compatible with detection in the presence of 3IAbRQSp/ (SEQ ID NO:9) ) was added to the complex. 2 μL of amplicon was combined with 18 μL of LbCas12a-gRNA complex in a black 384-well assay plate and fluorescence monitored using a Tecan plate reader.

(Table 20) Exemplary gRNA sequences for the detection of coronavirus

Example 62
Electrochemically Detectable Reporter Molecules for DETECTR Reactions This example describes reporter molecules with electrochemically detectable moieties for use in DETECTR reactions. Reporter molecules are ssDNA containing a modified thymine nucleobase conjugated to a ferrocene moiety and a fluorescent moiety (eg, fluorescein) conjugated to the 5' end via a phosphodiester bond. Reporter molecules can be biotinylated at the 3' end. The sequences of the two ssDNA reporter molecules are 5'-YXXTTATTXX-3' (SEQ ID NO: 391) and 5'-YXXTTATTATTXXZ-3' (SEQ ID NO: 392) , where X is ferrocene-labeled thymidine (Fig. 149). Panel A), Y is 6-carboxyfluorescein (Figure 149 panel B) and Z is the 3' biotin moiety (Figure 149 panel C).

The assay was performed using a 5'-YXXTTATTXX-3' (SEQ ID NO:391) reporter oligonucleotide, a programmable nuclease targeting HERC2, and a HERC2 target nucleic acid. Detection was performed using a DropSens μSTAT ECL instrument and a DropSens screen-printed carbon electrode. Aliquots of the DETECTR reaction were collected at multiple time points after its initiation.