WO2023057422A1

WO2023057422A1 - Universal buffer in methods for safe and rapid detection of pathogen infection

Info

Publication number: WO2023057422A1
Application number: PCT/EP2022/077522
Authority: WO
Inventors: Holger Summer; Stefan Golz; Kirsten LEINEWEBER; Katrin Zwirglmaier; Rosina EHMANN
Original assignee: Bayer Aktiengesellschaft
Priority date: 2021-10-06
Filing date: 2022-10-04
Publication date: 2023-04-13

Abstract

A lysis buffer comprising one non-ionic surfactant is provided which can be used as a one- step reagent of the inactivation, storage, amplification, and/or detection of pathogens and/or their nucleic acids and proteins. Various embodiments of the lysis buffer of the invention comprise other substances that are compatible or useful in inactivating pathogens, e.g. viruses, storing nucleic acids, amplifying nucleic acids, purifying nucleic acids, detecting nucleic acids, and/or other procedures for analysis of nucleic acids, as well as for the detection of antigens. Methods and kits based on the lysis buffer are also provided, including those for rapid lysis of cells and direct use of the resulting cell lysates in RT-PCR, LAMP and antigen detection.

Description

UNIVERSAL BUFFER IN METHODS FOR SAFE AND RAPID DETECTION OF

PATHOGEN INFECTION

FIELD

[001] The present disclosure relates to an improved method for inactivation of virus material in a biological sample optionally followed by storage, transport and detection of viruses in the biological sample using DNA or RNA analysis method as well as antigen testing.

BACKGROUND

[002] When handling biological samples in preparation for testing for the presence of pathogens, it is important to inactivate potential pathogens. In particular, viral particles need to be inactivated in order to prevent persons to get infected by handling the samples. Further, as the samples often are not tested directly but have to be transported from the site of sample taking to the laboratory in which the actual testing for the presence of the pathogen is performed, the biological sample and in particular the molecules which are to be detected have to be preserved from degradation. Yet further, the testing methods that test for the presence of characteristic molecules of a pathogen are prone to different ingredients that interfere with the test and foster wrong results. Nowadays, these different necessities for the different stages are addressed by using different buffers, i.e. a lysis buffer, a storage/transport buffer, and an analysis buffer.

[003] Some representative RT-PCR methods for detection of viral RNA require lysis of a biological sample in lysis buffer containing harsh chemicals under highly denaturing conditions to inactivate e.g. RNases and stabilize RNA followed by isolation of the RNA using e.g. column purification to remove these chemicals from the lysis buffer, which can interfere with the subsequent detection methods, such as RT-PCR. For example, the lysis buffers included in the standard TRIzol RNA preparation kits typically contain components such as guanidinium thiocyanate, phenol, and/or chloroform, which degrades or denatures proteins such as polymerases and would therefore interfere with the subsequent PCR reactions. CN 1 240 848 C for example discloses a method for detecting Pestivirus suis utilizing RT-PCR combined with ELISA technology to detect the amplified nucleic acid. However, the disclosed method also includes cumbersome purification steps because of the use of harmful reagents for nucleic acid extraction, comprising the use of TRIzol reagents.

[004] The majority of today’s pathogen detection methods include the use of two or more buffers to accomplish the above outlined necessities for lysis, stabilization during transport/storage and non-interference with the detection method to be used. The buffers are changed in time and money consuming purification steps.

[005] A time-consuming and costly step in SARS-CoV-2 diagnostics is RNA extraction from sample material. Similar to the virus transport media, delivery bottlenecks repeatedly occurred with the RNA extraction reagents in the course of the pandemic.

[006] At peak times in the course of the coronavirus pandemic to date, > 1 .5 million clinical samples per week have been tested for SARS-CoV-2 using PCR in Germany alone. The need for virus transport medium and RNA extraction kits was correspondingly high. The average cost of a tube with virus transport medium is around 1 euro. The cost of RNA extraction reagents is around EUR 3 to 10 per sample (based on currently available commercial RNA extraction kits), plus the personnel-associated costs.

[007] During the current SARS CoV-2 pandemic it became evident that rapid testing of samples at the point-of-care is needed to allow a fast assessment for the presence of a virus. Usually, the applied tests are lateral flow tests for the presence of antigens of the virus, such as the nucleocapsid protein. Only after a positive result in such lateral flow tests, further testing is indicated by DNA or RNA detection methods, e.g. PCR-based testing. However, as state of the art buffers are not compatible for both methods of testing without further ado, a further sample has to be taken, transported to a lab or stored until the test can take place. This is inefficacious and time consuming. This need for additional sampling also bears the danger that the titer or infection status of the subject to be tested has meanwhile changed. Further, during SARS CoV-2 pandemic there were temporary global bottlenecks in the availability of reagents and plastic goods that are essential for sampling and transport. In particular, the procurement of virus transport medium, which is required for the transport of samples, is associated with very long delivery delays.

[008] In addition to the gold standard of RT-PCR, rapid antigen tests are another cornerstone of virus diagnostics. A positive result in the rapid tests designed for point-of- care use, nowadays is usually confirmed with a PCR test. In order to avoid a second sampling for the confirmatory test, which is unpleasant for the patient and not always possible for logistical reasons, a buffer system that is suitable for both rapid tests and PCR would be advantageous.

[009] Accordingly, there is a need for the provision of a buffer and methods that allows for lysis of samples, inactivation of the virus, and that provide a lysate that can be directly and optionally in parallel be used in different detection methods, such as DNA/RNA analysis methods as well as methods to detect specific proteins and thereby providing for a universal buffer that makes purification steps superfluous and can be used in multiple methods.

[0010] Some disadvantages of these methods are the multiple steps, instability of analytes, lack of sensitivity, increased time, increased costs, and reagents needed to obtain results. A problem to be solved is also to provide for methods and reagents that provide an acceptable balance to address all of these needs.

[0011] The present invention provides methods and buffers that overcome the above drawbacks and are particularly suitable for applications in the point-of-care area, e.g. in low resource regions, and at the same time are compatible with applications in laboratorybased standard diagnostics, that eventually need further transportation.

[0012] A solution to this technical problem is provided by the embodiments characterized in the items and claims.

BRIEF SUMMARY

[0013] The present application provides compositions that are suitable for inactivation of the viruses or bacteria in a sample, stabilizing the analytes during transport and/or storage, and analysis of the samples for the presence of the analytes, preferably nucleic acids and/or antigens. The inventors have unexpectedly found that with the lysis buffer according to the present invention not only the cell lysis of e.g. human cells in a sample can be achieved, but that the contained pathogens like viruses are inactivated so that they are not infective anymore. This finding allows for the use of the lysis buffer for inactivating pathogens in a sample in order to facilitate subsequent handling and transportation. Further, the use of the buffer according to the present invention allows for the use of the lysate in several detection methods as disclosed herein without the need for purification steps. Moreover, the use of the buffer according to the present invention allows for storage and/or transportation of the lysate. Thereby, the present invention provides for a fast, save and reliable detection of pathogens, in particular viruses from biological samples.

[0014] Hence, the present application relates to the use of a lysis buffer according to the invention for inactivating pathogens, preferably viruses and/or bacteria, in a sample. Various embodiments of the lysis buffer of the invention comprise other substances that are compatible or useful in lysing cells and/or viruses, storing nucleic acids and/or proteins, amplifying nucleic acids, purifying nucleic acids, detecting nucleic acids and/or proteins, and/or other procedures for analysis such as sequencing of nucleic acids and/or proteins. The lysis buffer is considered a one-step reagent of the inactivation, preparation, storage, transport amplification, and/or detection of pathogens, in particular viruses, in samples.

[0015] Methods and kits based on the compositions are also provided, including those for rapid lysis of cells and inactivation of the pathogens, optional storage and/or transportation, and use of the resulting cell lysates in analysis for the presence of a pathogen in the sample by detecting virus specific DNA, RNA and/or antigen.

[0016] In particular, the present invention relates to a method to inactivate a pathogen, preferably an RNA virus or DNA virus, or a bacterium, comprising contacting at least one sample with a lysis buffer to produce a lysate, wherein the lysis buffer comprises a nonionic surfactant, glycerol, and at least one salt. The method of inactivating generally comprises contacting at least one cell and/or at least one virus with the lysis buffer according to the invention for a sufficient amount of time to cause the cell or virus to lyse and the virus to being inactivated. Additional optional steps may be included in the inactivation method.

[0017] It has surprisingly been found that a single buffer of the invention can be suitable for inactivating viruses, lysis of cells, viruses and bacteria, preserving the lysate and the analytes, by e.g. preventing bacterial and/or fungal growth, and can be present as a subcomponent of reactions leading to amplification and detection or sequencing of nucleic acids, or detection of antigens. Accordingly, the present invention provides a method for inactivating a virus in a sample. Further, it provides a method for preparing a sample which is suited for storage and/or transport. Further, the invention also provides for a method for detecting a virus in a sample which comprises all steps of lysing the cells in a sample to obtain a lysate, storing and/or transporting the sample to the place of analysis, and analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA and/or virus antigens in the lysate. For instance, the buffer of the invention can be suitable to inactivate viruses present in a sample, and to lyse a cell, such as a human, plant or animal cell, infected with a virus. The obtained lysate may be used as template in subsequent reactions leading to amplification, detection and quantification of nucleic acids or detection and/or quantification of antigens without further purification. The present invention furthermore relates to a method preparing a sample intended for the analysis for the presence of an RNA virus or DNA virus comprising

- inactivating the virus in at least one sample by the method for inactivating according to the invention to produce a lysate and thereby inactivating the virus; and

- storing and/or transporting the lysate.

The invention also relates to a method for detecting a virus in a sample comprising

- preparing the sample with the method for preparation according to the invention, and

- analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA or a virus antigen in the lysate.

[0018] The inventors moreover have surprisingly found that by using the buffer according to the present invention, detection of viral nucleic acids and virus antigens is possible without the need for purification and/or rebuffering. Accordingly, in a particular preferred embodiment, the present invention relates to a method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; taking one or more aliquot of the lysate; analyzing the one or more aliquot for the presence of a virus in the sample by detecting an antigen of the virus in the lysate; optionally storing and/or transporting the remainder of the lysate and/or a further aliquot thereof; and analyzing the remainder of the lysate or the further aliquot thereof for the presence of a virus in the sample by detecting virus DNA, and/or virus RNA in the lysate or the further aliquot thereof.

[0019] The present application also provides kits. In general, the kits contain the lysis buffer of the invention and two or more vessels for receiving an aliquot of a lysate. The present invention for the first time provides a method that allows multiple testing by DNA/RNA analysis and antigen testing without the need for changing buffers. Accordingly, the methods according to the invention may include the step of aliquoting the lysate to allow point-of-care testing and in addition lab testing from the same lysate. Accordingly, the kit according to the invention comprises two or more vessels for receiving an aliquot of the lysate. The kits can further comprise one or more substances, materials, reagents, etc. that can be used for lysis of cells or viruses, storage of nucleic acids or lysates, be present during amplification of nucleic acids, or detection or quantification of nucleic acids. In embodiments, some or all of the materials, reagents, etc. necessary to lyse cells or viruses, amplify nucleic acids, and/or detect and/or quantify nucleic acids are included in the kit.

[0020] The invention also relates to the use of a lysis buffer according to the present invention and kits according to the invention in a method of inactivating a virus in a sample comprising contacting a sample with the lysis buffer to produce a lysate and to inactivate the virus, transporting and/or storing the lysate, and analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA or a virus antigen in the lysate.

[0021] The invention also relates to the use of a lysis buffer according to the invention and kits in a method to detect an RNA or DNA virus comprising contacting a sample with the lysis buffer to produce a lysate and to inactivate the virus, taking one or more aliquot of the lysate, analyzing the lysate for the presence of a virus in the sample by detecting a virus antigen in the aliquot, storing and/or transporting the remainder of the lysate and/or one or more further aliquot, analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA in the remainder of the lysate and/or the one or more further aliquot. Preferably, in case of RNA virus detection reverse transcribing RNA within the remainder of the lysate and/or the one or more further aliquot to obtain cDNA, and amplifying at least one nucleic acid from an RNA virus in the remainder of the lysate and/or the one or more further aliquot using a set of primers derived from the RNA virus is also provided.

DETAILED DESCRIPTION

[0022] In the following different embodiments of components and steps of the methods and kits of the invention are disclosed and shall be understood to apply to all embodiments. Unless stated otherwise, any particular embodiment of a component or a step being declared as preferred shall be construed as being preferred for all aspects of the invention.

Lysis Buffer

[0023] The methods, kits and uses according to the invention comprise the (use of the) buffer according to the present invention (referred to as “the lysis buffer” or “lysis buffer according to the invention”). In the following preferred embodiments of the lysis buffer are disclosed and meant to apply to all embodiment of the present invention. [0024] In a preferred embodiment, the lysis buffer comprises at least one non-ionic surfactant, glycerol, and at least one salt.

[0025] In a preferred embodiment, the lysis buffer does not contain a guanidinium salt, more preferably the lysis buffer does not contain a guanidinium salt selected from the group consisting of guanidinium thiocyanate, guanidine hydrochloride, guanidine sulfate, guanidine acetate, guanidine nitrate, guanidine phosphate.

[0026] It has surprisingly been shown that the lysis buffer of the invention is suitable for inactivation of viruses and can be further used to store and/or transport of the lysate as well as being included as a component of reaction mixtures for amplification of nucleic acids by PCR methods, such as reverse transcription PCR (RT-PCR), and quantitative PCR (qPCR), as well as loop-mediated isothermal amplification (“LAMP”). In addition, the lysate can directly be used with antigen testing, such as lateral flow assays.

[0027] The lysis buffer comprises at least one detergent capable of disrupting membranes such as cell membranes and/or virus membranes.

[0028] In some embodiments, the lysis buffer comprises at least one non-ionic surfactant. In some embodiments, the at least one non-ionic surfactant has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group.

[0029] As used herein, the meaning of "surfactant" is the broadest definition that is readily recognized by a person of ordinary skill in the art. That is, surfactants are wetting agents that lower the surface tension of a liquid and/or lower the interfacial tension between two liquids. A surfactant that does not have a positive or negative charge in water, yet is soluble in water, is a "non-ionic surfactant". Combinations of two or more non-ionic surfactants are encompassed within the term "non-ionic surfactant".

[0030] Suitable non-ionic surfactants include, but are not limited to, Triton X-100, Igepal CA-630 (Nonidet P-40), Conco Nl, Dowfax 9N, Igepal CO, Makon, Neutronyx 600's, Nonipol NO, Plytergent B, Renex 600's, Solar NO, Sterox, Serfonic N, T-DET-N, Tergitol NP, Triton N, BIGCHAP (N,N-bis-(3-DGIuconamidopropyl)cholamide) or deoxy-BIGCHAP (N,N-bis(3- Gluconamidopropyl) deoxycholamide); Decanoyl-N-methylglucamide; n- Decyl a-DGIucopyranoside; n-Decyl [3-D-Glucopyranoside; n-Decyl [3-D-Maltopyranoside; Digitonin; n-Dodecyl [3-D-Glucopyranoside; n-Dodecyl a-D-Maltoside; n-Dodecyl |3-D- Maltoside; heptanoyl-N-methylglucamide; n-Heptyl [3-D-Glucopyranoside; N-Heptyl [3-D- Thioglucopyranoside; n-Hexyl [3-D-Glucopyranoside; 1 - Monooleoyl-rac-glycerol; Nonanoyl-N-methylglucamide; n-Nonyl a-D-Glucopyranoside; n-Nonyl [3-D- Glucopyranoside; Octanoyl-N-methylglucamide; n-Octyl a-D-glucopyranoside; n-Octyl [3- D-Glucopyranoside; Octyl [3-D-Thiogalactopyranoside; Octyl [3-D-Thioglucopyranoside; Polyoxyethylene Esters (such as 8-stearate polyoxyethylene ester (Myrj 45), 40-stearate polyoxyethylene ester (Myrj 52), 50-stearate polyoxyethylene ester (Myrj 53), and 100- stearate polyoxyethylene ester (Myrj 59)), Polyoxyethylene Ethers (such as those containing one or more ethyl groups, methyl groups, pentyl groups, cetyl groups, stearyl groups, oleyl groups, hexyl groups, octyl groups, decyl groups, lauryl groups, myristyl groups, heptyl groups, tridecyl groups, isohexadecyl groups, and combinations thereof); Polyoxyethylenesorbitan esters (such as those containing one or more monolaurate groups, monooleate groups, monopalmitate groups, monostearate groups, trioleate groups, and tristearate groups, and combinations thereof, including, but not limited to the "Tween" series of detergents); Sorbitan esters (such as those containing one or more monolaurate groups, monooleate groups, monopalmitate groups, monostearate groups, sesquioleate groups, trioleate groups, tristearate groups, and combinations thereof); Tergitol; n-Tetradecyl [3-D-Maltoside; the Triton series of detergents, including, but not necessarily limited to, Triton X-100 (t-Octylphenoxypolyethoxyethanol) and its derivatives, Triton X-114, Triton X-405, Triton X-101 , Triton N-42, Triton N-57, Triton N-60, Triton X- 15, Triton X-35, Triton X-45, Triton X-102, Triton X-155, Triton X-165, Triton X-207, Triton X-305, Triton X-705-70 and Triton B-1956; Nonylphenyl Polyethylene Glycol (Nonidet P- 40; NP-40, Igepal CA630); the Air Products series of Surfynol surfactants, including, but not necessarily limited to, Surfynol 104, Surfynol 420, Surfynol 440, Surfynol 465, Surfynol 485, Surfynol 504, Surfynol PSA series, Surfynol SE series, Dynol 604, Surfynol DF series, Surfynol CT series, and Surfynol EP series, for example Surfynol 104 series (104, 104A, 104BC, 104DPM, 104E, 104H, 104NP, 104PA, 104PG50, 104S), and Surfynol 2502; Tyloxapol; n-Undecyl [3-D-Glucopyranoside, and any non-ionic Octylphenol Ethoxylate surfactant. Additional non-limiting examples include the Dow Chemicals' Dowfax series of non-ionic surfactants, such as the N-series and the DP-series of surfactants, including, but not necessarily limited to, DOWFAX 63N10, DOWFAX 63N13, DOWFAX 63N30, DOWFAX 63N40, DOWFAX 81 N13, DOWFAX 81 N15, DOWFAX 92N20, DOWFAX 100N 15, DOWFAX EM-51 , DOWFAX 20A42, DOWFAX 20A64, DOWFAX 20A612, DOWFAX 20B102, DOWFAX DF-101 , DOWFAX DF-111 , DOWFAX DF-112, DOWFAX DF-113, DOWFAX DF-114, DOWFAX DF-117, DOWFAX WP-310, DOWFAX 50C15, DOWFAX DF-121 , DOWFAX DF-122, DOWFAX DF-133, DOWFAX DF-141 , DOWFAX DF-142, and DOWFAX DF-161. Yet other additional non-limiting examples include the pluronic series of surfactants from BASF, including but not limited to, 10R5, 17R2, 17R4, 25R2, 25R4, 31 R1 , F108 series, F127 series, F38 series, F68 series, F77 series, F87 series, F88 series, F98 series, L10, L101 , L121 , L31 , L34, L43, L44 series, L61 , L62 series, L64, L81 , L92, N-3, P103, P104, P105, P123, P65, P84, P85, and F127.

[0031] Any suitable amount of detergent and/or non-ionic surfactant may be included in the lysis buffer. In some embodiments, the lysis buffer comprises a non-ionic surfactant in an amount from about 0.05% to about 20%. In some embodiments, the lysis buffer comprises from about 0.05% to about 15%, from about 0.05% to about 10%, from about 0.05% to about 7%, from about 0.05% to about 5%, from about 0.05% to about 4%, from about 0.05% to about 3%, from about 0.05% to about 2%, from about 0.05% to about 1 %, or from about 0.05% to about 0.5%. In some embodiments, the lysis buffer comprises from about 0.1 % to about 15%, from about 0.1 % to about 10%, from about 0.1 % to about 7%, from about 0.1 % to about 5%, from about 0.1 % to about 4%, from about 0.1 % to about 3%, from about 0.1 % to about 2% or from about 0.1 % to about 1 %. In some embodiments, the lysis buffer comprises from about 0.5% to about 15%, from about 0.5% to about 10%, from about 0.5% to about 7%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, or from about 0.5% to about 2%. In some embodiments, the lysis buffer comprises from about 1 % to about 15%, from about 1 % to about 10%, from about 1 % to about 7%, from about 1 % to about 5%, from about 1 % to about 4%, from about 1 % to about 3%, or from about 1 % to about 2% of a non-ionic surfactant, such as Triton X-100. In some embodiments, the lysis buffer comprises from about 2% to about 15%, from about 2% to about 10%, from about 2% to about 7%, from about 2% to about 5%, from about 2% to about 4%, or from about 2% to about 3% of a non-ionic surfactant, such as Triton X-100. In some embodiments, the lysis buffer comprises from about 3% to about 15%, from about 3% to about 10%, from about 3% to about 7%, from about 3% to about 5%, or from about 3% to about 4% of a non-ionic surfactant, such as Triton X-100. In some embodiments, the lysis buffer comprises about 0.05%, about 0.1 %, about 0.5%, about 1 %, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11 %, about 12% about 13%, about 14%, or about 15% a non-ionic surfactant, such as Triton X-100. These percentages in case of fluidic non-ionic surfactants preferably refer to %(v/v). In case of non-ionic surfactants that are non-fluidic, e.g. in powder form, the percentages preferably refer to %(w/v). In a preferred embodiment, the lysis buffer comprises a non-ionic surfactant is Triton X-100 in an amount from about 0.05%(v/v) to about 20%(v/v), preferably 0.3%(v/v) to 3.5%(v/v), more preferably 0.5%(v/v) to 3%(v/v).

[0032] Although the lysis buffer may comprise one or more other components, and those components are not limited by the exemplary components disclosed herein, the lysis buffer will typically contain a solvent, such as water, an organic solvent, such as glycerol, or both. Although it is preferred that the solvent used be as pure as possible or practicable, solvents of any purity may be used. Thus, where water is included in the lysis buffer, it may be distilled water, double-distilled water, de-ionized water, sterilized water, or any combination thereof. The solvent, be it water or any other solvent or combination of water and any other solvent, may be treated before use to reduce or eliminate one or more chemical or biochemical activities, such as, but not limited to nuclease (e.g., RNase, DNase) activities. For example, the water or any other solvent or combination of water and any other solvent may be RNase-free. Likewise, the lysis buffer may be treated with sterilization techniques or with chemicals or biologicals, etc. to sterilize the composition or to reduce or eliminate one or more undesirable chemical or biochemical activities (e.g., RNase, DNase, etc.). For example, the lysis buffer may contain an RNase inhibitor.

[0033] The lysis buffer of the invention may also comprise one or more salts, such as a sodium salt, a potassium salt, a magnesium salt, a manganese salt, a zinc salt, a cobalt salt, or a combination of two or more of these salts. In a preferred embodiment, the salt is selected from the group consisting of disodium phosphate, sodium chloride, magnesium chloride, manganese chloride, and potassium chloride. The salts may be added in any suitable amount and for any reason, including, but not limited to, as an aid in lysis of cells or viruses, for moderation of surfactant cloud point and foam level, and for improved function of reagents involved in amplification of nucleic acids. In a preferred embodiment, the lysis buffer comprises disodium phosphate in a concentration from about 1 mM to about 50 mM, preferably 3 mM to 30 mM, more preferably 4 mM to 25 mM.

[0034] The above components comprised in the lysis buffer reflect the core and gist of the invention. It will be understood by those of ordinary skills in the art that the buffer is an aqueous solution containing the components as outlined herein. However, further components may be added in accordance with then necessities, e.g. preventing RNase activity, or maintaining a certain pH. The lysis buffer of the invention may also comprise one or more buffers suitable for inclusion in nucleic acid amplification, such as, but not limited to Tris-HCl. The lysis buffer of the invention may also comprise one or more reducing agents. Reducing agents help to break bonds (such as disulfide bonds) which loosen the secondary structure of RNA and can facilitate reverse transcriptase (RT) enzyme initiation of transcription. Any suitable reducing agent may be included in the lysis buffer, such as dithiothreitol (DTT). The lysis buffer in a preferred embodiment further comprises one or more of the following: Tris-HCl, dithiothreitol (DTT), RNase-free water, RNase inhibitor, and mixtures thereof.

[0035] The lysis buffer may be adjusted to a suitable pH. In a preferred embodiment, the pH of the lysis buffer is from about 6.5 to about 8.5 or from about 7.0 to about 8.5. For example, the pH of the lysis buffer can be from about 7.5 to about 8.5 or from about 7.5 to about 8.0. In some embodiments, the pH of the lysis buffer is about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, or about 8.5. Preferred pH for any embodiment of the invention is a pH of about 7.5 to about 8.5, more preferably from about 7.6 to 8.9, most preferably a pH of about 7.8.

[0036] In a particularly preferred embodiment the lysis buffer comprises: about 0.5%(v/v) to 3%(v/v) Triton-X 100, about 1 ,5%(v/v) to about 10%(v/v) glycerol, about 0.3 mM to about 2mM DTT, about 4mM to about 25mM Na2HPO4, from about 4 mM to about 25 mM Tris HCI; and RNAse-free water, and has a pH of about 7.8; wherein the lysis buffer does not contain a guanidinium salt. In a further preferred embodiment the lysis buffer consists of the listed components and optionally comprises one or more RNase- inhibitor.

[0037] In a preferred embodiment the lysis buffer in the actually used composition comprises buffer comprises: about 0.5%(v/v) Triton-X 100, about 1.7%(v/v) glycerol, about 0.3 mM DTT, about 4.2 mM Na2HPO4, about 4.2 mM Tris HCI; and RNAse-free water, and has a pH of about 7.8; wherein the lysis buffer does not contain a guanidinium salt. In a further preferred embodiment, the lysis buffer consists of the listed components and optionally comprises one or more RNase-inhibitor.

[0038] The skilled person understands that a buffer may be provided in a concentrated form. To this end, the lysis buffer according to the invention may also be provided as a composition comprising multiples of the concentrations as given in above preferably used composition (stock solution), such as for examples a 2-fold to 100-fold stock solution, preferably a 3-fold to 50-fold, more preferably a 4-fold to 10-fold. For example, concentrated stock solutions can also be formulated as a 100-fold stock solution, 50-fold stock solution, 20-fold stock solution, 10-fold stock solution, 6-fold stock solution 5-fold stock solution, or 2-fold stock solution. In a most preferred embodiment, the stock solution is a 6-fold stock solution. In a particular preferred embodiment, the stock solution of the lysis buffer is 6-fold stock solution and comprises: about 3%(v/v) Triton-X 100, about 10%(v/v) glycerol, about 2 mM DTT, about 25 mM Na2HPO4, from about 25 mM Tris HCI; and RNAse-free water, and has a pH of about 7.8; wherein the lysis buffer does not contain a guanidinium salt. In a further preferred embodiment the stock solution of the lysis buffer is 6-fold stock solution and consists of the listed components and optionally comprises one or more RNase-inhibitor.

[0039] The skilled person will acknowledge that such stock solution is provided in a concentrated form and is intended for dilution, e.g. by water, preferably RNase-free water, in an extend to result in a 1 -fold concentrated buffer. 1 -fold concentrated buffer preferably refers to a buffer with the components and concentrations as given in the previous paragraph. [0040] The lysis buffer of the invention may be made in advance, and optionally diluted before use. The lysis buffer may be diluted in any suitable diluent that does not interfere with the subsequent analysis of RNA, DNA and/or antigen. In some embodiments, the lysis buffer is diluted with water before use. In other embodiments, the lysis buffer is diluted with a medium. The lysis buffer can be diluted in any suitable ratio. For example, the lysis buffer may be diluted 100:1 , 50:1 , 25:1 , 20:1 , 10:1 , 5:1 , 4:1 , 3:1 , 2:1 , 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :15, 1 :20, 1 :25, 1 :30, 1 :40, 1 :50, or 1 :100 (lysis bufferdiluent).

[0041] In an aspect, the lysis buffer of the invention may be a concentrated stock comprising 1 %(v/v) to 5%(v/v) Triton X-100 and one or more of the following components: 5%(v/v) to 20%(v/v) glycerol, 0.5-4 mM DTT, 10-50 mM Na2HPO4, and 10-50 mM Tris- HCI. In some embodiments, the lysis buffer of the invention may be a concentrated stock comprising about 3%(v/v) Triton X-100 and one or more of the following components: about 10%(v/v) glycerol, about 2 mM DTT, about 25 mM Na2HPO4, and about 25 mM Tris- HCI. The pH of the concentrate stock may be from about 7.5 to about 8.0, preferably about 7.8.

[0042] The concentrated stock may be diluted with a diluent, such as RNase-free water, prior to use. In some embodiments, the concentrated stock may be diluted from 1 :15 to 1 :1 with RNase-free water prior to use. In some embodiments, the concentrated stock may be diluted 1 :6 with RNase-free water prior to use.

[0043] In some embodiments, RNase inhibitor to the lysis buffer is added prior to use, in particular if the virus is an RNA virus and the RNA is to be analysed. In some embodiments, the RNase inhibitor is added to the lysis buffer in an amount from about 1 : 100 to about 1 : 10000 prior to use. In some embodiments, the RNase inhibitor is added to the lysis buffer in an amount of about 1 :1000 prior to use. The skilled person will acknowledge that the RNase inhibitor may be added from a concentrated RNase inhibitor stock solution. The amount of RNase inhibitor stock solution to be added may be dependent on amount of RNase inhibitor contained in said stock solution. Amount of RNase inhibitor preferably refers to its activity which is known to the skilled person. A unit RNase inhibitor is preferably defined as the amount of RNase Inhibitor required to inhibit the activity of 5 ng of RNase A by 50%. Activity is measured by the inhibition of hydrolysis of cytidine 2, 3 -cyclic monophosphate by RNase A (J Biol Chem. 1979 Dec 25;254(24): 12484-7).

[0044] . In particular the activity is given as units per pl (ll/pl). In a preferred embodiment, RNase inhibitor is added to the lysis buffer prior to use in an amount to give a final concentration of 0.1 ll/pl to 40 ll/pl, preferably 0.2 ll/pl to 10 ll/pl, more preferably 0.3 ll/pl to 0.5 ll/pl, most preferred is a final concentration of the RNase inhibitor of 0.4 ll/pl. In a particularly preferred embodiment, a solution comprising 40 ll/pl RNase inhibitor is added in an amount of 1 :1000 into the lysis buffer prior to use. In an embodiment the final concentration of RNase inhibitor is 0.2U/pl to 0.26U/pl

Pathogens and Samples

[0045] The methods, kits and uses according to the invention relate to lysing a sample, inactivation of viruses in the sample optionally followed by transport and/or storage of the lysate and analysis of the lysate. Further, the Examples show that by using the lysis buffer according to the invention, bacterial pathogens can be inactivated. In the following preferred embodiments of the pathogens are disclosed and meant to apply to all embodiment of the present invention.

[0046] The Examples as provided herein exemplify the invention by providing data for the SARS CoV-2. The skilled person will acknowledge that the technical effect of the invention is not restricted to this particular virus or variants thereof. The skilled person will recognize that the invention is instantly workable for any virus, such as for DNA viruses and RNA viruses. In particular embodiments, the virus is an enveloped virus. Enveloped viruses are viruses which outermost layer are typically derived from portions of the host cell membranes and contain phospholipids and proteins in addition to optionally included viral glycoproteins. Preferably, the virus is selected from the group of enveloped viruses consisting herpesviridae, poxviriridae, hepadnaviridae, asfarviridae, flaviviridae, alphaviridae, togaviridae, coronaviridae, kolmioviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, bunyviridae, filoviridae, retroviridae, and coronaviruses. In particular embodiments, the virus is an enveloped RNA virus, preferably a coronavirus, more preferably SARS CoV-2 or a variant thereof. [0047] The virus may be an animal or plant pathogenic virus, in some embodiments the virus is a plant pathogenic virus selected from the group consisting of Tobacco mosaic tobamovirus (TMV), Tomato spotted wilt tospovirus (TSWV), Tomato yellow leaf curl begomovirus (TYLCV), Cucumber mosaic cucumovirus (CMV), Potato virus Y (potyvirus, PVY), Cauliflower mosaic caulimovirus (CaMV), African cassava mosaic begomovirus (ACMV), Plum pox potyvirus (PPV), Brome mosaic bromovirus (BMV), Potato virus X (potexvirus, PVX), Rice tungro disease agents RTBV, Rice tungro spherical virus (RTSV), Rice yellow mottle (RYMV), Rice hojablanca virus (RHBV), Barley yellow dwarf luteoviruses (BYDV), Maize streakmastrevirus (MSV), Maize rayado fino virus (MRFV), Maize dwarfmosaic virus, Maize sugarcane mosaic virus, Sweet potato feathery mottle potyvirus(SPFMV), sweet potato sunken vein closterovirus (SPSVV), African cassava mosaic disease (ACMD) begomovirus complex, Banana bunchy top nanovirus (BBTV), Banana streak badnavirus (BSV), Barley yellow dwarf disease luteovirus complex, Cucumber mosaic cucumovirus, Maize streak mastrevirus (MSV), Maize dwarf mosaic/Sugarcane mosaic potyviruses, Rice tungro disease complex, Rice yellow mottle sobemovirus (RYMV), and Sweet potato feathery mottle potyvirus (SPFMV). The skilled person will acknowledge that the present invention is particularly suited for animal and/or human pathogenic viruses. Such viruses have extensively been reviewed (Viral Pathogens of Domestic Animals and Their Impact on Biology, Medicine and Agriculture Encyclopedia of Microbiology. 2009: 805-819). Preferred animal or human pathogenic viruses are selected from the group consisting of African horse sickness virus; African swine fever virus; BCG, Bacille Calmette-Guerin; bluetongue virus; camelpox virus; classical swine fever virus; endogenous JSRV-related retroviruses; foot-and-mouth disease virus; goatpox virus; human immunodeficiency virus; human T cell leukemia virus; jaagsiekte sheep retrovirus; low virulence NDV; Maedi-visna virus; Newcastle disease viruses; OIE, Office International des Epizooties; RSV, Rous sarcoma virus; RT-PCR, reverse transcriptase-polymerase chain reaction; SA, sialic acids; SARS, severe acute respiratory syndrome; SARS-CoV, SARS-Coronavirus; sheeppox virus; transmissible spongiform encephalopathy; variola virus; virulent NDV; Marburg virus; Ebola virus; Hanta virus; and influnenza A virus, and influenza B virus. [0048] It has also been shown, that the lysis buffer according to the invention also inhibits growth of pathogenic bacteria. Hence, in one embodiment the pathogen according to the invention is a bacterial pathogen, preferably human, animal or plant pathogenic bacteria, preferably human pathogenic bacteria, more preferably selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, and Yersinia pestis. Accordingly, in one particular embodiment, the present invention relates to the use of a lysis buffer according to the invention for inactivating bacteria in a sample, preferably for inactivating a bacterium selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, and Yersinia pestis, most preferred for inactivating Bacillus anthracis in a sample. Further, the invention also relates to method to inactivate a pathogenic bacterium, preferably Bacillus anthracis in a sample, comprising contacting at least one sample thought to contain said pathogenic bacterium with a lysis buffer to produce a lysate, wherein the lysis buffer comprises a non-ionic surfactant, glycerol, and at least one salt.

[0049] The methods and uses herein comprise the step of contacting a sample with the lysis buffer according to the invention. “Sample” in connection with the present invention refers to any type of sample which are thought to contain a virus. In a preferred embodiment the sample is thought to contain a DNA virus or RNA virus, more preferably an enveloped DNA virus or enveloped RNA virus. The sample can be an environmental sample, a sample taken from a plant or an animal or a human being. In a preferred embodiment, the sample contains at least one cell that is thought to be infected with a DNA virus or a RNA virus, preferably an enveloped DNA virus or enveloped RNA virus

[0050] Preferably, the sample according to the invention is a biological sample. A biological sample includes cells from a subject, for example a biological sample may be blood, tissue, urine, salvia, lavage, feces etc. from one or more subjects. In some embodiments, the at least one cell or at least one virus is present in a biological sample. It will be understood by those of skills in the art that the biological sample may be taken directly from the one or more subjects or may be taken from “indirect” origin. For instance, if the presence of virus in a population is to be tested or quantified, samples could be taken from a source that has or had contact with the subjects of the population, e.g. wastewater from the sewerage or sewage treatment plant could be taken to analyze the presence of a virus in feces or urine contained in the wastewater. [0051] In a preferred embodiment, the biological sample is collected using a swab. For example, the swab can be used to collect a biological sample from any mucous membrane such as, nose, cheek, pharynx, or mouth. In an aspect, the swab may be used to collect a sample from any place the cells of interest, for example epithelial cells, are located. In a particular embodiment, the biological sample is a nasal swab. In another embodiment, the biological sample is selected from the group consisting of a bodily fluid (nasopharyngeal aspirate, sputum, saliva, urine, blood, etc), excrements, lavage, and tissue. The swap may also be used to take samples from other sources, such as environmental samples or samples taken from wastewater. The at least one cell contained in the sample can be any eukaryotic or bacterial cell. Preferably the at least one cell is a eukaryotic cell. The cell can be any cell of interest, including, but not limited to, mammalian cells, avian cells, amphibian cells, reptile cells, insect cells, and plant cells. For example, the cell can be a human cell, a monkey cell, a rat cell, a mouse cell, a dog cell, a cat cell, a pig cell, a horse cell, a hamster cell, a rabbit cell, a frog cell, an insect cell, etc.. In some embodiments, the cell is an epithelial cell. Particularly preferred are human cells, more preferred human epithelial cells collected in a sample taken with a swab.

[0052] By "at least one cell", it is meant not only a single cell, but a single cell type. Thus, two or more cells can mean not only two or more cells of the same cell type, but one or more cell of two different cell types. Unless otherwise specifically noted, it is not relevant whether a population of a single cell type is present or a population of two or more cell types is present. Regardless, the methods of the invention (including those discussed below) will provide the stated effects.

[0053] Furthermore, the terms "at least one cell" and "a cell" are, unless otherwise noted, used interchangeably herein to define a single cell, a collection of a single type of cell, or a collection of multiple types of cells, at least one cell of each type being present.

[0054] Similarly, the terms "at least one virus" and "a virus" are, unless otherwise noted, used interchangeably herein to define a single virus, a collection of a single type of virus, or a collection of multiple types of viruses, at least one virus of each type being present. [0055] The biological sample in an embodiment is a sample of a plant. The skilled person will understand that the present invention is also suited for detecting plant pathogens, in particular plant pathogenic viruses. In such case, the sample is preferably a plant sample, such as a leaf sample, a stem sample, or a root sample, preferably the biological sample of a plant contains at least one plant cell.

Methods

Inactivation Method

[0056] In a first aspect, the present invention provides a method to inactivate an RNA or DNA virus comprising contacting at least one sample with a lysis buffer according to the invention to produce a lysate.

[0057] The method comprises contacting at least one sample with a lysis buffer according to the invention for a sufficient amount of time to cause the cell and/or virus to lyse. In an embodiment, the method of to inactivate an RNA or DNA virus comprises adding the lysis buffer of the invention to least one sample. The method optionally further comprises incubating the mixture of the lysis buffer and the sample for a time and at a temperature sufficient for lysing the cells and/or viruses contained in the sample, preferably for 5 minutes at room temperature.

[0058] “Inactivation” in connection with viruses is to be understood as modifying and/or lysing virus particles within a sample as to they are not able to infect a cell or organism. Viral inactivation renders viruses unable to infect. In a preferred embodiment, inactivated refers to less than 10⁶ PFU/ml in the lysate, more preferably less than 10⁴ preferably PFU/ml, yet more preferably less than 10³ PFU/ml, most preferred less than 8*10² PFU/ml.

[0059] Previous methods also included the storage by any suitable means prior to lysis. The means for storage can vary depending on the type of sample and length of storage. For example, biological samples can be stored at room temperature (e.g., 22-25°C), placed on ice, or refrigerated (e.g., 4°C) for short-term storage or can be frozen for longterm storage. Frozen biological samples can be stored for example at -20°C, -80°C or in liquid nitrogen. [0060] In prior art methods, the sample, such as a biological sample, is often placed in a transport or storage medium for storage. This bears the danger of storing potentially hazardous material that could lead to infections of the handling personnel. However, such storage of the sample is made superfluous, as the present invention provides method for inactivating the virus so that it is not infectious anymore. This provides safety in storage and transport.

[0061] Further, a method for preparing a sample is provided that includes the storage and/or transport of the lysate. This is made possible by the unexpected finding that the lysis buffer inhibits bacterial and yeast growth and thereby protects the lysate during storage and/or transportation.

[0062] The amount of time the sample, optionally comprising at least one cell and/or virus, is contacted with the lysis buffer of the invention may vary and can be determined by one of skill in the art. In some embodiments, the amount of time the sample is contacted with the lysis buffer of the invention is an amount of time sufficient to cause lysis of at least one cell or virus. Due to the chemical nature of the lysis reaction, it is envisioned that the time can be quite short, such as 1 second or less. However, the time need not be so limited. Indeed, because the lysis buffer can be present during subsequent storage analysis for the presence of nucleic acids or antigens, the lysis time can be relatively long. Suitable times can range from 1 second or less to minutes, hours, or days. Exemplary times for contact include, but are not limited to, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 90 seconds, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes or more.

[0063] In various embodiments, additional optional steps are included in the inactivation method. It has been demonstrated that viral inactivation will occur without additional steps by use of the lysis buffer according to the invention. However, to expedite inactivation, or viruses can be exposed to one or more mechanical disruption techniques. Any known mechanical disruption technique may be used, including, but not limited to, vortexing, repeated pipetting, inversion, shaking, and stirring. Thus, inactivation can be accomplished, at least in part, by homogenizing. Depending on the ultimate use of the lysate, the mechanical techniques, when used, may be applied gently to minimize shearing stresses on the nucleic acids or antigens. In some embodiments, lysis can be accomplished, at least in part, through the action of biological or biochemical substances.

[0064] Any suitable volume of lysis buffer may be used in the method of the invention and can be varied depending on the sample. In some embodiments, a volume of about 50 pl, about 100 pl, about 200 pl or more of lysis buffer is used. In some embodiments, a volume of 100 pl of lysis buffer is used.

[0065] Freezing of the lysate can improve virus inactivation. Thus, in some embodiments, the lysate may optionally be frozen. The lysate may be frozen once or more than once. In other words, the lysate may be subjected to one or multiple freeze-thaw cycles. Any suitable conditions may be used to freeze the lysates. It will be understood that temperature and duration can be optimized depending on various factors, such as the size and type of the sample and amount of lysate. In some embodiments, the lysate is incubated at a temperature of -20°C or less. For example, the lysate may be incubated at a temperature selected from, but not limited to, about -20°C, about -80°C, or about - 120°C prior to analysis. In some embodiments, the lysates are placed on dry ice prior to analysis. The lysate may be incubated as described above for any suitable amount of time. In some embodiments, the lysate may be incubated at a temperature of -20°C or less for at least about 10 minutes. For example, the lysate may be incubated at a temperature of -20°C or less for about 10 minutes, about 30 minutes, about 1 hour, or about 24 hours, or more. In some embodiments, the lysates are incubated at a temperature of -80°C for 10 minutes.

[0066] The inactivation method can also include one or more steps that result in separation of cell and/or viral components from other cell and/or viral components. Thus, for example, the method may comprise centrifuging the lysate to remove not lysed cells and/or viruses, cell and/or viral membranes and proteins from nucleic acids. While not preferred, it can also include precipitation of one or more cellular or viral component from others, for example, by the addition of one or more salts, organic solvents (e.g., alcohol), or through heat treatment and subsequent centrifugation. Other techniques for separating cellular and/or viral components from each other are known to those of skill in the art, and any suitable technique may be used, each being selected based on the desired outcome. Selection and performance of an appropriate technique is well within the skill level of those of skill in the art.

[0067] Preferably, the method to inactivate an RNA virus or DNA virus, preferably an enveloped RNA virus or an enveloped DNA virus, in at least one biological comprises mixing the biological sample with the lysis buffer of the invention, wherein the biological sample is a swab, such as a nasal swab comprising at least one cell, such as an epithelial cell and/or at least one virus, such as a coronavirus. The method to inactivate in an embodiment further comprises squeezing the nasal swab to release the cells and/or virus particles into the lysis buffer. In some embodiments, the method to inactivate further comprises incubating the mixture of the lysis buffer and the biological sample for 5 minutes at room temperature to produce a lysate. In an embodiment, the method further comprises centrifuging the lysate, for example at 12,000 rpm for 2 minutes at room temperature and collecting the supernatant.

Preparation Method

[0068] After inactivation of the viruses in the sample, the lysate may be stored and transported. It has surprisingly been found that the lysis buffer inhibits bacterial and yeast growth. Further, it has been proven in the examples herein, that the inactivation of the viruses in the sample is accomplished so that the lysate does not contain any measurable plaque forming units (PFU) of the virus. Accordingly, the present invention provides for a superior method for preparing a sample intended for the analysis for the presence of an RNA or DNA virus. In particular, the present finding shows that storing and/or transporting of the lysate can be included in the method without the need for further precautionary measures to prevent infections of persons in contact with the lysate, or bacterial or yeast overgrowth. Hence, the invention relates to a method preparing a sample intended for the analysis for the presence of an RNA virus or DNA virus comprising inactivating the virus in at least one sample by the method to inactivate an RNA virus or DNA virus according to the invention to produce a lysate and thereby inactivating the virus and storing and/or transporting the lysate. As the first step of the method for preparing a sample intended for the presence of an RNA virus or DNA virus refers to the method to inactivate an RNA virus or DNA virus according to the invention, any embodiment of that method also applies to the respective step of the method for preparing a sample according to the invention.

[0069] The method for preparing a sample includes storing and/or transporting the lysate for a period of time before use. In a preferred embodiment the sample is stored and/or transported for 2 hours to 1 week, preferably 1 to 5 days.

[0070] The lysate can be stored at any number of temperatures; in particular, as it inhibits bacterial and yeast growth. For example, it can be stored at relatively high temperatures (e.g., 37°C), at room temperature (e.g., 22°C - 25°C), in the refrigerator (e.g., 4°C), frozen (e.g., -20°C), or deep frozen (e.g., -80°C or lower). Preferably storing and/or transporting is conducted at a temperature between -80°C and 37°C, more preferably between -80°C and room temperature, yet more preferred between -20°C and room temperature, most preferred between 4°C and room temperature.

[0071] The lysis method may also include manipulation of one or more lysate component. Thus, the method may include purification of one or more nucleic acid from the lysate and/or amplification of one or more nucleic acid from the lysate. Various embodiments of the method of lysis include any and all procedures that are known for use with lysates.

Detection Method

[0072] The inventors have unexpectedly found that by the use of the lysis buffer according to the invention it is possible to perform all stages from inactivation of the virus, storage and/or transporting as well as a variety of analysing methods. Accordingly, in a particular preferred embodiment the invention relates to a method for detecting a virus in a sample comprising preparing the sample with the method for preparing a sample intended for the analysis for the presence of an RNA virus or DNA virus; and analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA or an antigen in the lysate.

[0073] The method for detecting a virus in the sample, hence, comprises the steps of the method of preparing a sample intended for the analysis for the presence of an RNA virus or DNA virus. Accordingly, any embodiment of the method for preparing and the therein comprised method to inactivate an RNA virus or DNA virus applies to any embodiment of the method for detecting a virus a sample.

[0074] In a particular preferred embodiment, the invention relates to a method for detecting a virus in a sample comprising contacting said sample with a lysis buffer according to the invention to produce a lysate and to inactivate the virus; taking one or more aliquot of the lysate; analyzing an aliquot for the presence of a virus in the sample by detecting an antigen of the virus in the lysate; optionally storing and/or transporting the remainder of the lysate and/or one or more further aliquot thereof; and analyzing the remainder of the lysate and/or the one or more further aliquot thereof for the presence of a virus in the sample by detecting virus DNA, virus RNA in the lysate or the further aliquot thereof.

[0075] The inventors have found that the lysis buffer according to the invention does allow for different types of analysis methods, such as nucleic acid detection methods as well as antigen testing. It has further been found that by using the lysis buffer, the need for a purification step to get rid of any buffer substances potentially interfering with the respective method of analysis. Accordingly, the methods according to the invention do not comprise an RNA, DNA and/or protein extraction step. In other words, the lysate is used in the method of analysis without an or any extraction step. In case of a nucleic acid-based analysis method, such as PCR, qPCR, RT-PCR, RT-qPCR, or loop-mediated isothermal amplification (“LAMP”) and sequencing, the lysate is used as a template in such methods. In case of a test for the presence of viral antigens, the lysate may directly be applied, such as for example on a lateral flow test assay. Nevertheless, if applicable, the lysate may also be diluted with the appropriate buffer for the respective method for analysis.

[0076] The present invention further provides for the possibility analyse the same lysate for the presence of both, virus nucleic acids and antigens. The skilled person will understand that analysis is performed with the lysate as produced in the step of inactivating the virus. Analyzing the lysate may be performed by taking an aliquot from the lysate as needed and storing or transporting the remainder for further analysis. As the skilled person will acknowledge, this for the first time offers the possibility to perform point- of-care testing, e.g. through a rapid antigen test, and a remote, optionally lab-based PCR based test, from a single sample. Accordingly, the step of analyzing the lysate in a preferred embodiment comprises the step taking an aliquot of the lysate and analyzing the aliquot for the presence of a virus in the sample by detecting virus DNA, virus RNA or a protein antigen in the aliquot.

[0077] The invention hence relates to a method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; taking one or more aliquot of the lysate; analyzing the one or more aliquot for the presence of a virus in the sample by detecting virus DNA, virus RNA or a virus antigen in the aliquot, and storing and/or transporting the remainder of the lysate. The skilled person will also acknowledge that such aliquoting may take place either before storing and/or transporting the lysate or after. Accordingly, the invention also relates to a method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; storing and/or transporting the lysate; taking one or more aliquot of the stored and/or transported lysate; and analyzing the one or more aliquot for the presence of a virus in the sample by detecting virus DNA, virus RNA or an antigen in the one or more aliquot.

[0078] In particularly preferred embodiment, the method for detecting a virus in a sample does not comprise a nucleic acid extraction step, preferably the method does not contain an RNA extraction step.

[0079] “Aliquot” in connection with the present invention means a part of the lysate Aliquot or aliquot part denotes the portion of the lysate to be analysed if the entire sample cannot or should not be completely examined. Dividing the lysate allows the same sample to be used multiple times without impairing the quality, e.g. through repeated thawing/freezing. In connection with the present invention aliquot is to be understood as being any (any number, any size) partial portions of a lysate, irrespective whether the quotient of the total sample and the sample amount is an integer (e.g. 10 ml of a 100 ml sample) or a non-integer (e.g. for 15 ml of a 100 ml sample). Taking the aliquot may refer to dividing the lysate in two or more aliquots or taking only a part of the lysate as the “aliquot”.

[0080] In preferred embodiment, the invention relates to a method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; taking one or more aliquot of the lysate; analyzing the one or more aliquot for the presence of a virus in the sample by detecting an antigen of the virus in the lysate; storing and/or transporting the remainder of the lysate or a further aliquot thereof; and analyzing the remainder of the lysate or the further aliquot thereof for the presence of a virus in the sample by detecting virus DNA, or virus RNA in the lysate or the further aliquot thereof. The skilled person will also acknowledge that such aliquoting may take place either before storing and/or transporting the lysate or after. Accordingly, the invention also relates to a method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; storing and/or transporting the lysate; taking one or more aliquot of the lysate; analyzing the one or more aliquot for the presence of a virus in the sample by detecting an antigen of the virus in the lysate; and analyzing the lysate or a further aliquot thereof for the presence of a virus in the sample by detecting virus DNA, or virus RNA in the lysate or the further aliquot thereof. In a particular preferred embodiment, the method comprises the step of taking two aliquots, wherein a first aliquot is analyzed the presence of a virus in the sample by detecting an antigen of the virus in the first aliquot, and wherein the second aliquot is analyzed for the presence of a virus in the sample by detecting virus DNA, or virus RNA. In a particular preferred embodiment the lysate, i.e. including the remainder of the lysate and the aliquots are used directly in the analyzing steps, preferably without any purification steps, in particular without DNA, RNA or protein extraction.

[0081] In an aspect, the invention relates to methods of amplifying, identifying, detecting, quantifying and/or analyzing a target nucleic acid and a virus antigen, comprising, for example: (a) obtaining a sample, optionally comprising no cells or one or more cells, for example epithelial cells;

(b) contacting the sample with a lysis buffer of the invention to produce a lysate;

(c) taking an aliquot of the lysate and analysing the lysate aliquot thereof by detecting a virus antigen;

(d) optionally freezing the remainder of the lysate;

(e) thawing the remainder of the lysate, if necessary, and centrifuging the remainder of the lysate, if necessary;

(f) collecting and transferring the supernatant to a new reaction vessel, such as a reaction tube or microtiter plate, if centrifugation is necessary

(g) optionally freezing the supernatant,

(h) thawing the supernatant, if necessary, and

(i) amplifying, identifying, detecting and/or analysing a target nucleic acid by any suitable means.

[0082] In some embodiments, the amplifying, identifying, detecting, and/or analysing a target nucleic acid is carried out using a PCR technique, such as, for example, PCR, qPCR, RT-PCR, RT-qPCR, or LAMP. Methods for PCR and LAMP are well known, and any suitable method can be used in the methods of the invention. In particular embodiments, the method does not comprise an RNA extraction step.

[0083] In particular embodiments, the biological sample is collected using a swab.

[0084] In particular embodiments, the biological sample is a nasal swab.

[0085] In particular embodiments, the at least one sample comprises at least one human cell.

[0086] In particular embodiments, the method further comprises incubating the mixture of the lysis buffer and the sample for 5 minutes at room temperature to produce a lysate.

[0087] In particular embodiments, the method further comprises centrifuging the lysate and collecting the supernatant, wherein the supernatant is taken as the lysate in subsequent steps and the one or more aliquot is taken from the supernatant. [0088] In particular embodiments, the lysate is centrifuged at 12,000 rpm for 2 minutes at room temperature.

Analysis methods

[0089] Methods herein comprise the step of analysing the lysate or an aliquot thereof by detecting virus DNA, virus RNA or a virus antigen. The skilled person will understand that analysing the lysate may be conducted as necessary and known to those skilled in the art. They include nucleic acid based and/or antibody-based technologies, such as point-of-care lateral flow assays.

Nucleic acid detection

[0090] Preferred methods of detecting virus DNA and/or virus RNA in the lysate and/or an aliquot thereof are PCR, qPCR, RT-PCR, RT-qPCR, loop-mediated isothermal amplification (“LAMP”), or sequencing methods. As the skilled person will acknowledge, targets for such analysis may be chosen in accordance with the particular needs. In particular, the skilled person may choose sequences within the genome of a virus to provide for both, sensitivity and specificity. Specific sequences may be amplified, detected or sequenced using suited primers directed to the specific sequences or flanking regions. In particular embodiments, the set of primers is directed to a viral RNA gene, preferably a coronavirus gene, more preferably a SARS CoV-2 gene, selected from the group consisting of an E gene, an N gene, and an RdRP gene.

[0091] The skilled person will acknowledge that the type of nucleic acid detection may be selected according to the needs. In particular, depending on the type of virus to be detected, e.g. a RNA virus or a DNA virus. Further, it may be desirable to characterize the virus contained in the sample, e.g. its RNA or DNA sequence. Accordingly, the term “detecting virus DNA or virus RNA” is to be understood as comprising methods that give information about the presence of the DNA or RNA, its amount and/or its sequence, and hence include amplification methods such as PCR; qPCR, RT-PCR, RT-qPCR, and LAMP as well as methods for sequencing the DNA or RNA.

[0092] In a preferred embodiment, the present invention relates to a method for the identification of a subject infected with SARS CoV-2 or a variant of SARS CoV-2 comprising inactivating the biological sample by contacting the biological sample with a lysis buffer according to the invention to produce a lysate, taking one or more aliquot of the lysate; analysing an aliquot for the presence of an SARS CoV-2 antigen, preferably using a lateral flow test, and reverse transcribing RNA from a further aliquot of the lysate or the remainder of the lysate to obtain cDNA, and subjecting the cDNA to PCR assay using a set of primers derived from a nucleotide sequence of the SARS CoV-2 genome or the genome of the variant of SARS CoV-2.

[0093] In a preferred embodiment, the set of primers is directed to the E gene and comprise or consist of SEQ ID NO: 2 and/or SEQ ID NO: 3, or a complement thereof.

[0094] In a preferred embodiment, the set of primers is directed to the RdRP gene and comprise or consist of SEQ ID NO: 5 and/or SEQ ID NO: 6, or a complement thereof.

[0095] In particular embodiments, said further aliquot of the lysate or the remainder of the lysate is directly used in the reverse transcription.

[0096] In one embodiment the present invention relates to a method for the identification of a subject infected with SARS CoV-2 or a variant of SARS CoV-2 comprising inactivating the biological sample by contacting the biological sample with a lysis buffer according to the invention to produce a lysate, taking two aliquots of the lysate; analysing a first aliquot for the presence of an SARS CoV-2 antigen, preferably using a lateral flow test, and reverse transcribing RNA from the second aliquot to obtain cDNA, and subjecting the cDNA to PCR assay using a set of primers derived from a nucleotide sequence of the SARS CoV-2 genome or the genome of the variant of SARS CoV-2.

[0097] The method may also comprise three analysis steps, i.e. analyzing an aliquot for the presence of an SARS CoV-2 antigen, analysing a second aliquot by reverse transcribing RNA from the second aliquot to obtain cDNA, and subjecting the cDNA to PCR assay using a set of primers derived from a nucleotide sequence of the SARS CoV- 2 genome or the genome of the variant of SARS CoV-2; and sequencing the virus RNA by suited sequencing methods. [0098] For the analysis of the lysate or an aliquot by detecting virus RNA or virus DNA, mastermixes can be used. In one embodiment, any suitable PCR or LAMP MasterMix can be used. The term “MasterMix” refers to a premixed solution of all reagents and essential components required for the PCR or LAMP reaction, except for the analyte, i.e. the lysate or the aliquot of the lysate. Several commercial PCR or LAMP MasterMix formulations, including qPCR MasterMix formulations, are available and can be used in the instant invention. Alternatively, a PCR or LAMP MasterMix can be created by mixing components necessary for a PCR or LAMP reaction to occur, such as a DNA polymerase, nucleotides, and magnesium, and optionally a reverse transcriptase and RNase inhibitor. In some embodiments, the PCR or LAMP MasterMix and the lysate are added in a 5:1 ratio (PCR MasterMix: lysate).

[0099] In an aspect, the qPCR is a one-step RT-PCR using a LightCycler® 480 QPCR reader (Roche). However, any suitable PCR machine may be used in the methods of the invention.

[00100] In an aspect, the data are analysed using LightCycler® 480 software (Roche). However, any suitable software may be used to analyse the PCR data.

[00101] In an aspect, the assays described herein are sensitive enough to detect about 20 copies or less, about 10 copies or less, about 5 copies or less, about 4 copies or less, about 3 copies or less, or about 2 copies or less of a target nucleic acid within a sample.

[00102] If necessary, the nucleic acids, i.e. the virus DNA or virus RNA, can be detected by amplification or can be amplified before the actual detection. Numerous techniques for amplification of nucleic acids are known and widely practiced in the art, and any of those techniques are applicable according to the method of this invention. One of skill in the art may select the amplification method based on any number of considerations, including, but not limited to, speed, sensitivity, usefulness in amplifying a particular type of nucleic acid (e.g., RNA vs. DNA), and reliability.

[00103] Although the method may comprise isolation or purification (to at least some extent) of nucleic acids, it has surprisingly been discovered that amplification of target nucleic acids may be accomplished without purification of the nucleic acid beforehand. Thus, the lysate or the respective aliquot of the invention is suitable for direct nucleic acid amplification. In some embodiments, amplifying is by a PCR or LAMP technique. In certain embodiments, the PCR technique is qPCR or RT-PCR (including RT-qPCR).

[00104] Loop-mediated isothermal amplification (LAMP) is a method well known to those skilled in the art. It is - compared to e.g. PCR or RT-PCR - a resources saving method without the need of well-equipped laboratories. In LAMP the target sequence is amplified at a constant temperature of 60 to 65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. An additional pair of "loop primers" can further accelerate the reaction. The amount of DNA produced in LAMP is considerably higher than PCR-based amplification. The actual amplification product can be detected via photometry, measuring the turbidity caused by magnesium pyrophosphate precipitate in solution as a byproduct of amplification. This allows easy visualization by the naked eye or via simple photometric detection approaches for small volumes. The reaction can be followed in real-time either by measuring the turbidity or by fluorescence using intercalating dyes, such as SYTO 9. Dyes, such as SYBR green, can be used to create a visible color change that can be seen with the naked eye without the need for expensive equipment, or for a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated with the number of copies initially present. Hence, LAMP may also be used for quantification. In-tube detection of LAMP DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis. Another method for visual detection of the LAMP amplicons by the unaided eye was based on their ability to hybridize with complementary gold-bound ss-DNA and thus prevent the normal red to purple-blue color change that would otherwise occur during salt-induced aggregation of the gold particles. So, a LAMP method combined with amplicon detection by AuNP can have advantages over other methods in terms of reduced assay time, amplicon confirmation by hybridization and use of simpler equipment (i.e., no need for a thermocycler, electrophoresis equipment or a UV trans-illuminator). For different viruses commercially available kits for LAMP can be used, such as Loopamp System from Eiken Chem (LMC403) or Eazyplex system from Amplex Diagnostics (Cat. no. 7680) for detecting SARS CoV-2

[00105] Where one or more RNA are the nucleic acids of interest, the method may comprise a cDNA synthesis prior to, or at the time of, amplification. Numerous cDNA synthesis protocols are known in the art, and any suitable protocol may be used. For example, the qScript XLT reverse transcriptase from Quantabio may be used to prepare cDNA from RNA templates.

[00106] It has been shown that the methods of the invention are particularly well-suited for use to detect viral RNA or viral DNA within mammalian biological samples, such as a nasal swab, or non-biological samples.

[00107] The methods of the invention provide several advantages over conventional viral RNA or viral DNA detection or analysis assays. For example, the method of the invention avoids the need for an RNA or DNA extraction step, thereby reducing labor and making the method of the invention faster. RNA extraction kits, such as QIAamp® DSP Viral RNA Mini kit (Qiagen) which is currently required by the Centers for Disease Control and Prevention (CDC) in the United States for COVID-19 testing, generally contain columns to separate and purify nucleic acids from components that may interfere with amplification methods. Therefore, the elimination of an RNA extraction step also results is less material consumption since no columns are required. Furthermore, reagents necessary for RNA extraction are not always readily available, thus, elimination of these reagents increases availability of the assays. This can be of particular importance during pandemics, for example, when a high volume of test samples needs to be analysed and rapid results obtained. The provision of a high-throughput, rapid method to detect viral RNA that is both precise and sensitive is a significant advantage.

[00108] The method of the invention preferably comprises using the lysate or an aliquot thereof directly for RT-PCR (e.g., one-step RT-qPCR) or LAMP. The steps of detecting virus DNA or virus RNA preferably is performed without an RNA or DNA extraction step.

[00109] In some embodiments, one or more control reactions are included, such as a control reaction to permit normalization of the amount of nucleic acid being amplified with respect to other amplification reactions that are being performed concurrently, or with respect to a standard amplification curve. Such control reactions can comprise adding one or more exogenous nucleic acids to the reaction and performing an amplification on that nucleic acid. The control reaction can alternatively comprise amplifying sequences present in nucleic acids naturally present in the cell or biological sample of interest, where such sequences have a known copy number and amplification efficiency. In other embodiments, control reactions for known sequences are performed in reaction vessels separate from the reaction vessel in which the amplification of interest is being performed. Various other control reactions are known and widely used for amplification reactions, and any of those control reactions may be included in the method of the present invention to determine the success and efficiency of one or more steps in the amplification process.

[00110] In some embodiments, the control reaction is an internal control that permits the practitioner to evaluate, and thus normalize if desired, the number of cells present in a particular cell lysate sample. In this way, conclusions about the amount of target nucleic acid (e.g., a particular viral RNA or DNA gene) in a sample may be made. More specifically, when comparing amplification results of two different samples, it is often not possible to determine with a high degree of accuracy, the number of cells or virus in the original sample, the number of cells or virus successfully lysed, or the total amount of nucleic acid liberated from each sample. Thus, accurate comparisons of the total amount of a target nucleic acid in different samples is not possible. Currently, housekeeping genes or rRNA (ribosomal RNA) species are used as markers to standardize or normalize samples from different cells or tissues. However, the currently used internal standards have been reported to be inconsistent, and thus do not provide the accuracy and repeatability that is needed for an internal control.

[oom] An internal control that is standardized among different samples and cell types is thus a desirable feature of a PCR or LAMP protocol. In certain embodiments, the present invention encompasses such an internal control by including within the compositions, methods, and kits, primers that are specific for unique sequences on one or more of the chromosomes of a given cell or virus. Because these unique genomic sequences are present in only one copy per haploid genome (i.e., two copies per cell), they can be used to prepare a standard curve for a particular amplification procedure. Inclusion of the primers in the amplification reactions for test samples (either in the same reaction vessel or in a second reaction vessel comprising the same components) results in an amplification curve for the unique genomic sequences. These curves can be compared to the standard curve for each primer set, and the amount of nucleic acid, and thus the number of cells or viruses in the original sample, can be calculated. With this knowledge, the amount of a target nucleic acid in numerous different samples can be determined, and accurately compared with other samples.

Primers

[00112] Any suitable primers can be used in the methods of the invention. Suitable primers can readily be identified by those of skill in the art depending upon the target nucleic acid of interest to be identified. Several primers useful to identify common nucleic acids of interest are commercially available and can be used in the methods of the invention.

[00113] In some embodiments, the primers are directed to an RNA viral genome, such as the SARS CoV-2 genome or to the genome of SARS CoV-2 variants. The complete SARS CoV-2 genomic sequence can be found in GenBank Accession No. NM 908947.3. Kits for the detection of SARS CoV-2 containing primers are commercially available from various vendors. It is contemplated that such primers can be used in the method of the invention. In some embodiments, the primers are directed to the E gene, the N gene, or the RdRP gene. In some embodiments, the primers are directed to the E gene and comprise or consist of SEQ ID NO: 2 and/or SEQ ID NO: 3, or a complement thereof. In some embodiments, the primers are directed to the RdRP gene and comprise or consist of SEQ ID NO: 5 and/or SEQ ID NO: 6, or a complement thereof.

Probes

[00114] Any suitable nucleic acid probes can be used in the method of the invention. Suitable nucleic acid probes can readily be identified by those of skill in the art depending upon the target nucleic acid of interest to be identified. Several probes useful to identify common nucleic acids of interest are commercially available and can be used in the methods of the invention. [00115] In some embodiments, the nucleic acid probes are directed to an RNA or DNA viral genome, such as the SARS CoV-2 genome the genome of a SARS CoV-2 variant. Kits for the detection of SARS CoV-2 containing nucleic acid probes are commercially available from various vendors. It is contemplated that such probes can be used in the method of the invention. In some embodiments, the nucleic acid probes are directed to the E gene, the N gene, or the RdRP gene. In some embodiments, the nucleic acid probe is directed to the E gene and comprises or consists of SEQ ID NO: 1 , or a complement thereof. In some embodiments, the nucleic acid probe is directed to the RdRP gene and comprises or consists of SEQ ID NO: 4, or a complement thereof.

[00116] In some embodiments, the probes can be tagged at 5’ and/or 3’ end, for instance with a fluorophore and/or a quencher. The tag can be any suitable tag, such as a HEX tag or a BHQ1 tag.

[00117] The RT-qPCR protocol described herein is based on standard diagnostic TaqMan-based RT-qPCR methods for SARS CoV-2. In principle, the protocol can be varied, such as, for example, other viral genes could be measured, different primer and probe sequences, different master mixes, quenchers, fluorophores, apparatuses, etc could be used. Other RT-qPCR methods such as SYBR® Green based methods can also be used in the method of the invention.

Sequencing methods

[00118] As outlined herein, detection of virus RNA or virus DNA may also be conducted using suited sequencing methods as known to the skilled person. Such sequencing methods include but are not limited to Maxam-Gilbert sequencing, next generation sequencing methods, Chain-termination methods, Large-scale sequencing and de novo sequencing, Shotgun sequencing, Single molecule real time (SMRT) sequencing, Nanopore DNA sequencing, Massively parallel signature sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, Combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, or Microfluidic System sequencing. Antiqen detection

[00119] An “antigen” is a molecule or molecular structure, that may be present on the outside of a virus, and that can be bound by an antigen-specific. Antigens are proteins, peptides (amino acid chains) and polysaccharides (chains of monosaccharides/simple sugars), or lipids and nucleic acids combined with proteins and polysaccharides. Antigens are "targeted" by antibodies. Antibodies are produced to match an antigen to bind it after they have come into contact with it; this allows a precise identification or matching of the antigen. Accordingly, antigen detection is preferably performed using methods employing antigen-specific antibodies. Hence, analysing the lysate or aliquots for the presence of an virus antigen is preferably performed using immunoassays, such as dipstick lAs, immunochromatographic assays or fluorescence immunoassays. Preferably, the analysis for the presence of a virus antigen is used as a “point-of-care” test, such as immunochromatographic assay which gives visual results that can be seen with the naked eye, preferably a lateral flow assay. Lateral flow assays are well known in the art and inter alia reviewed in Koczula KM, Gallotta A. (Lateral flow assays. Essays Biochem. 2016;60(1 ):111-120. doi:10.1042/EBC20150012; which is incorporated herein by reference).

[00120] Any lateral flow test may be applied as needed. In preferred embodiments, the lateral flow test is a commercially available one. In particular embodiments, the virus to be detected is SARS CoV-2-virus and the commercially available

[00121] In particular embodiments, the lysate or aliquots are directly used in the reverse transcription and analysis for the presence of a virus antigen.

[00122] In some embodiments, the method of the invention can be used to detect an RNA or DNA virus using the lysis buffer described herein. For example, the method of the invention can be used to detect an RNA or DNA virus such as, but not limited to, togavirus, coronavirus, retrovirus, picornavirus, calicivirus, reovirus, orthomyxovirus, paramyxovirus, rhabdovirus, buynavirus, arenavirus, or fibovirus. In some embodiments, the RNA virus is a coronavirus such as, but not limited to, corona SARS viruses, such as SARS CoV or SARS CoV-2 or variants of those viruses and corona MERS viruses and variants of those viruses. In a particular embodiment, the method of the invention can be used to detect SARS CoV-2.

[00123] Thus, the present invention also relates to a method for the identification of a subject infected with SARS CoV-2 comprising obtaining a lysate from a biological sample obtained from the subject, reverse transcribing the RNA within the lysate to obtain cDNA, and subjecting the cDNA to PCR assay using a set of primers derived from a nucleotide sequence of the SARS CoV-2 genome.

Kits

[00124] Kits are also provided. In general, the kits contain the lysis buffer of the invention and two or more vessels for aliquoting a lysate. The kits can further comprise one or more substances, materials, reagents, etc. that can be used for lysis of cells or viruses, storage of nucleic acids or cell lysates, or manipulation or analysis of nucleic acids, such as amplification of nucleic acids. In some embodiments, some or all of the materials, reagents, etc., necessary to lyse cells or viruses, amplify nucleic acids, and/or purify nucleic acids are included in the kit.

[00125] For example, a kit may contain a container holding the lysis buffer of the invention, and, in the same or a separate container, at least one reagent for amplification of a target nucleic acid. Thus, it can comprise at least one primer, such as two primers, for amplification of a target nucleic acid. It also may include at least one other primer for amplification of a target nucleic acid, which can be, but is not necessarily, the same nucleic acid (and even the same sequence within the same nucleic acid) that is the target for one or more other primer(s) in the kit. In some embodiments, the kits comprise two or more primers for amplifying one or more unique genomic sequences.

[00126] A kit of the present invention may be a kit for analysis of nucleic acids, further comprising a lysis buffer according to the invention and two or more vessels for aliquotation of a lysate. Hence, the method also relates to a kit comprising a lysis buffer, and two or more vessels for aliquotation of a lysate; wherein the lysis buffer comprises at least one non-ionic surfactant, glycerol, and at least one salt. [00127] In particular embodiments, the kit further comprises a rapid antigen test, preferably a lateral flow test.

[00128] In particular embodiments, the kit further comprises at least one reagent for amplification of a target nucleic acid.

[00129] In particular embodiments, the kit comprises at least one primer and/or at least one probe for amplification of a target nucleic acid.

[00130] In particular embodiments, the kit is a kit for detection of RNA using one-step RT-qPCR.

[00131] In particular embodiments, the kit comprises one or more of at least one reverse transcriptase, at least one DNA polymerase, an RNase inhibitor, nucleotides, primers, probes, labels, or any combination thereof.

[00132] In particular embodiments, the target nucleic acid is derived from a RNA virus.

[00133] In particular embodiments, the RNA virus is a coronavirus.

[00134] In particular embodiments, the RNA virus is SARS CoV-2.

[00135] In particular embodiments, the kit comprises a set of primers selected from SEQ ID NO: 2 and/or SEQ ID NO: 3, or a complement thereof and SEQ ID NO: 5 and/or SEQ ID NO: 6, or a complement thereof, optionally the kit further comprises a probe of SEQ ID NO:1 or a complement thereof and/or a probe of SEQ ID NO: 4 or a complement thereof.

[00136] For example, it can be a kit for detection of RNA using a one-step RT-qPCR, further comprising at least one container containing the lysis buffer of the invention. Such kits can comprise, in packaged combination, at least one reverse transcriptase, at least one DNA polymerase, such as Taq DNA polymerase, Pfu DNA polymerase, Pfx DNA polymerase, Tli DNA polymerase, Tfl DNA polymerase, and klenow, an RNase inhibitor, nucleotides (e.g., any or all of the four common deoxynucleotides), primers, probes, or labels (such as, for example, SYBR green), or any combination of two or more of these. Alternatively, it can be a kit that can be used for detection of RNA using a two-step RT- qPCR. Alternatively, the kit can be one that can be used for detection of DNA using a PCR technique, such as qPCR. In addition, the kit can be one that is used for detection of short interfering RNA (siRNA). In some embodiments, the kits comprise transfection or transformation reagents.

[00137] The kits can comprise the components in a single package or in more than one package within the same kit. Where more than one package is included within a kit, each package can independently contain a single component or multiple components, in any suitable combination. As used herein, a combination of two or more packages or containers in a single kit is referred to as "in packaged combination". The kits and containers within the kits can be fabricated with any known material. For example, the kits themselves can be made of a plastic material or cardboard. The containers that hold the components can be, for example, a plastic material or glass. Different containers within one kit can be made of different materials. In embodiments, the kit can contain another kit within it. For example, the kit of the invention can comprise a kit for purifying nucleic acids.

[00138] The kit of the invention can comprise one or more components useful for amplifying target sequences. In embodiments, some or all of the reagents and supplies necessary for performing PCR are included in the kit. In some embodiments, some or all reagents and supplies for performing qPCR are included in the kit. In other embodiments, some or all reagents and supplies for performing RT-PCR are included in the kit. Nonlimiting examples of reagents are buffers (e.g., a buffer containing Tris, HEPES, and the like), salts, and a template dependent nucleic acid extending enzyme (such as a thermostable enzyme, such as Taq polymerase), a buffer suitable for activity of the enzyme, and additional reagents needed by the enzyme, such as dNTPs, dllTP, and/or a UDG enzyme. A non-limiting example of supplies is reaction vessels (e.g., microfuge tubes).

[00139] The kit can comprise at least one dye for detecting nucleic acids, including, but not limited to, dsDNA. In embodiments, the kit comprises a sequence-non-specific dye that detects dsDNA, such as SYBR® Green dye (Molecular Probes, Eugene, OR). The dye is preferably contained alone in a container. In embodiments, the dye is provided as a concentrated stock solution, for example, as a 50X solution. In embodiments, the kit comprises a passive reference dye. In these embodiments, the passive reference dye can be included in the kit alone in a separate container. The passive reference dye can be provided as a concentrated stock solution, for example, as a 1 mM stock solution. A nonexclusive exemplary passive reference dye is ROX dye. In embodiments, the kit contains either a DNA-detecting dye or a passive reference dye. In other embodiments, the kit contains both a DNA-detecting dye and a passive reference dye.

[00140] The invention, in general, is suitable for use in both research and diagnostics. That is, the compositions and methods of the invention can be used for the purpose of identifying various nucleic acids or expressed genes, or for other research purposes. Likewise, the compositions and methods can be used to diagnose numerous diseases or disorders of humans and animals. In addition, they can be used to identify diseased or otherwise tainted food products (e.g., foods that are infected with one or more pathogenic organisms), or the presence of toxic substances or toxin-producing organisms in a sample. Thus, the compositions and methods have human health and veterinary applications, as well as food testing and homeland security applications.

[00141] The present invention may be better understood by reference to the following examples, which are not intended to limit the scope of the claims.

FIGURE LEGEND

Figure 1 : Documentation of plaque test results, a) Plaque assay: virus with buffer B. b) Plaque assay: reference virus without treatment

Figure 2: Effect of buffer B on growth of various organisms

Figure 3: Effect of buffer B on growth of B. anthracis spores.

Figure 4: Effects of buffer B on the PCR amplification (ct values and curve shape)

Figure 5: Results of the eazyplex SARS-CoV-2 assay with a swab sample a) B-buffer treatment b) according to the manufacturer's protocol. The buffer treatment leads to shorter detection times.

Figure 6: Graphic representation of the different detection times with the Loopamp system Figure 7: Graphical representation of the data from Table 15

Figure 8: Sample photos of the different line intensities with I without treatment of the sample with buffer B for a virus concentration of 10³ PFU/ml

Figure 9: Graphic representation of the ct values of RT-PCR reactions using SARS CoV-2 virus culture diluted in buffer B or diluted in PBS and prior RNA extraction as a template

EXAMPLES

MATERIALS AND METHODS

Composition of PBS Buffer per 5 L aqueous solution:

40.00 g NaCI

1.00 g KCI

0.60 g KH2PO4

4.55 g Na₂HPO₄

LB medium plates were obtained from Merck (Cat. No. 1.102.830.500).

LB liquid medium were obtained from Merck (Cat. No. 1005.470.500.

The Examples provided herein below were made with an exemplary buffer according to the invention, referred to as “buffer B”.

Composition of a 6-fold concentrated stock solution of a lysis buffer according to the invention (referred to in the Examples as buffer B):

3 % Triton-X 100

10 % Glycerol

2 mM DTT

25 mM Na₂HPO₄

25 mM Tris HCI pH 7.8

Stored at 4°C

If not stated otherwise buffer B is used in 1-fold concentration, i.e. 1 :6 dilution of above’s composition in RNase-free water. For use in nucleic acid-based analysis the RNase-inhibitor (40 U/p I) was added in a final concentration of 0.1 l 1 100 pl (i.e. 0.4 U/p I) sample volume at day of use.

EXAMPLE 1

INACTIVATION CAPACITY

A) Inactivation capacity for SARS CoV2 virus particles

The extent to which buffer B inactivated SARS CoV2 virus particles was evaluated in a plaque test. This point is highly relevant for health and safety reasons, as the protective measures required for further sample processing are required if the buffer does not sufficiently inactivate the virus, i.e. leads to its non-infectiveness.

Plaque test

In the plaque test, infectious and replicable virus are detected by infecting cells, with subsequent overlaying by a highly viscous media. Formation of plaque-shaped foci indicate virus infection of a cell. Using different dilution levels, allows for quantification the number of infectious virus particles in the starting solution (plaque-forming-unit “PFU). The inactivation capacity of a substance can be precisely quantified by incubation with a reference virus stock solution and subsequent evaluation in the plaque test. However, the prerequisite is that the substance is not toxic to cells. To this end, toxic components was conducted by centrifugation with a cut-off of 3 kDa. Virus particles were held back, and the buffer passed through. The virus was resuspended in the same volume of culture medium.

Vero-E6 (ATCC no. CRL1586) cells were used as indicator cells for the plaque test. The plaque test was carried out in 24-well plates with 10 dilution series, in each case in duplicate for each batch. The reference virus stock was titrated with and without centrifugation (monitoring of virus loss via the column) and a batch of the reference virus solution was m ixed 1 : 1 with 2-fold buffer B (to a final concentration of 1 -fold buffer B) (also with and without centrifugation). After 1 hour of incubation, the inoculum was removed, and the cells were covered with viscous medium. After three days infection was stopped by fixation with formaldehyde, the cell lawn was stained, and the plaque formation was evaluated.

Results

Buffer B proved to be highly toxic to cells with complete detachment of the cell lawn in the undiluted starting solution and in a 10’¹ dilution. In the 10’² dilution, an impairment of the cell lawn can be seen after 1 hour of incubation, but the cells recover. Unfortunately, the corresponding cell-toxic components cannot be completely separated by the centrifugation column. Even the smallest of buffer residues in the column (a few drops) retain the high cell toxicity after resuspension. The plaque test could hence only be evaluated from a dilution level of 10’² on. At this dilution, no more plaques can be detected in two independent biological replicates. With an initial titer of 2.4*10⁶ PFU/ml, a reduction in infectivity by at least a factor of 10⁴ (i.e. 99.99%) can be demonstrated; see Figure 1 . A higher factor or complete inactivation can be assumed, but not technically detectable.

Titer determination

Virus titer was determined as follows:

Plaque mean value (from duplicate per dilution level) x 4 (dilution factor inoculum per 24 wells (250 pl) in relation to final unit per ml) x 2 (dilution factor virus in buffer or cell culture medium) = number of PFU/ml (plaque forming units):

Effect of centrifugation on reference virus stock’s titer

Table 1: Read-out reference virus stock (without centrifugation)

(₂*4*36*10⁴ + 2*4*1 .5*10⁵)/2 PFU/ml

= (288*10⁴ + 12*10⁵)/2 PFU/ml

= (28.8*10⁵ + 12*10⁵)/2 PFU/ml

= 20.4*10⁵ PFU/ml

= 2.4*10⁶ PFU/ml

Table 2: Read-out reference virus stock (with centrifugation)

(2*4*19*10⁴ + 2*4*1.5*10⁵ + 2*4*0.5*10⁶)/3 PFU/ml

= (152*10⁴ + 12*10⁵ + 4*10⁶)/3 PFU/ml

= (15.2*10⁵ + 12*10⁵ + 4*10⁶)/3 PFU/ml

= (1 .52*10⁶ + 1 .2*10⁶ + 4*10⁶)/3 PFU/ml

= 2.24*10⁶ PFU/ml

In conclusion, the titer of the reference virus stock before and after centrifugation is comparable. Hardly any virus loss through centrifugation was observed. Effect of buffer B on reference virus stock’s titer

Table 3: Read-out reference virus stock treated with buffer B (without centrifugation)

n/d: not detectable

Table 4: Read-out reference virus stock treated with buffer B (with centrifugation)

n/d: not detectable

Conclusion

The titer in both cases was below detection level, i.e. below 8*10² PFU/ml (that would be the first countable value for exactly one plaque in 10’² dilution).

Taking into account the minimum possible titer for buffer B (less than 8*10² PFU/ml) and the determined initial titer of the reference stock (2.4*10⁶ PFU/ml), buffer B results in a reduction of infectious units of at least about 10⁴ (factor 1 : 10,000).

B) Effect of buffer B on other groups of organisms - gram-positive and gramnegative bacteria as well as yeasts

As part of diagnostics, clinical samples are often stored for hours or days in the diagnostic laboratory due to longer transport routes or sample backlogs, often at room temperature or 4°C. When using common, non-inactivating sample transport solutions such as UTM (Copan) or saline solution, an overgrowth of the sample by oral flora or other contaminants can often be observed. This can inhibit virus detection or lead to false positive results. Hence, a buffer for sample storage or transportation should at least partially inhibit bacterial or yeast growth.

To investigate the respective inhibition by a buffer according to the invention, it was tested to what extent the B-buffer can inhibit the growth of different groups of organisms. Representatives of gram-positive bacteria (Bacillus anthracis, Staphylococcus aureus), gram-negative bacteria (Escherichia coli, Yersinia pestis) and yeasts (Candida albicans) were tested.

To this end, a colony from an overnight culture on LB medium plates of the respective organism was resuspended in 100 pl PBS buffer. 20 pl of this were put into tubes with 2 ml LB liquid medium + 400 pl buffer B 6-fold (corresponds to a final concentration of 1- fold buffer B) or, as a control, in 2 ml LB medium + 400 pl PBS and overnight at 37 °C incubated. The next day, the growth in the tubes with B-buffer or control tubes was compared using an ODeoonm measurement (Biophotometer, Eppendorf)

Further, the effect of buffer B on B. anthracis spores was subsequently tested. To this end, 50 pl of a spore suspension in dilutions of 10⁶, 10⁵ and 10⁴ spores/ml were given to 2 ml LB medium + 400 pl buffer B (6-fold; final concentration of 1-fold buffer B) or as a control in 2 ml LB medium + 400 pl PBS and incubated shaking overnight at 37°C.

The next day, the growth in the tubes with buffer B or control tubes was compared using an ODeoonm measurement (Biophotometer, Eppendorf).

Results

Table 5: growth inhibition properties of buffer B for various organisms

For B. anthracis, buffer B showed an almost complete inhibition of growth (Table 5 and Figure 2). Also, for C. albicans, S. aureus and Y. pestis buffer B showed growth inhibiting properties (Table 5 and Figure 2).

Table 6: growth inhibition properties of buffer B on B. anthracis spores

Treatment with buffer B showed complete inhibition of the growth of B. anthracis across all three dilutions tested (Table 6 and Figure 3). Even after incubation for a further 48 hours at 37 ° C (test for delayed germination of spores), no growth was detectable in the tubes treated with buffer B. Summary

Buffer B shows nearly complete growth inhibition of B. anthracis, both as vegetative cells and as spores. Growth inhibition was also found for other bacteria and yeast.

EXAMPLE 2

RT-qPCR

The gold standard of RNA virus diagnostics, like for SARS CoV2, is still RT-PCR. In standard laboratory diagnostics, a time-consuming and resource-intensive RNA extraction from the sample usually takes place before the PCR.

An RNA extraction takes about 1 to 2 hours, depending on the method, costs about 3 to 10 Euros and requires (depending on the method) various devices such as centrifuges, thermo blocks or automated purification devices.

By treating the sample with the buffer B, RNA extraction would be superfluous, and the sample could be used directly in the PCR. To demonstrate this, the following experiments were conducted.

Method

Evaluation whether buffer B has an inhibitory effect on the PCR reaction

Extracted genomic RNA from SARS CoV-2 was diluted in buffer B. At the same time, the RNA was diluted in H2O as a control. 5 pl of each dilution step was used in duplicate for the PCR.

Evaluation whether SARS-CoV-2 can be detected in clinical samples lysed in buffer B without RNA extraction.

A panel of 50 SARS CoV-2 positive clinical swab specimens was a) used for RNA extraction using QIAGEN Viral RNA Kit (Cat no. 52904) b) used directly in the PCR without further treatment c) mixed with buffer B and subsequently used in the PCR

5 pl of the resulting solution were used as template in each case.

Table 7: Reaction setup for PCR

5x OS Buffer: Tris CI, KCI, (NH4)2SO4, 12.5 mM MgCI₂, DTT; pH 8. 7 (20 °C) (source: QIAGEN-OneStep-RT-PCR-Kit-Handbook)

OS Enzyme Mix: 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.5% (v/v) Nonidet P-40, 0.5 % (v/v) Tween®20, 50% glycerol (v/v), stabilizer; pH 9. 0 (20°C) (source: QIAGEN- OneStep-RT-PCR-Kit-Handbook) Table 8: PCR Program for device platform MIC (Biomolecular Systems)

Further, a native SARS CoV-2 virus culture was used for confirming these results. To this end, two experimental setups were performed to compare direct use of lysate with previous RNA extraction.

The SARS CoV-2 virus culture was dilute either

1 . in buffer B, or

2. PBS, 140 pl of which was extracted with the QIAamp Viral RNA Kit according to the manufacturer's protocol, eluted in 60 pl.

10 pl of 1 . and 5pl of 2. were used as the template in the PCR

These experiments were carried out with Qiagen OneStep reagents on the MIC according to the above’s protocol. Results are given in Figure 9.

Results

Table 9: Effects of buffer B on the PCR amplification (ct values)

Using extracted SARS CoV-2 RNA it could be shown that the addition of buffer has no inhibitory effect on the PCR. There was no significant difference in the ct values or the course of the curve in a direct comparison with and without buffer B (Table 9, Figure 4).

Table 10: Comparison of the ct values in a PCR reaction from extracted RNA, without extraction and without extraction but with buffer B

Table 11: Evaluation of the data from Table 6, staggered according to ct value

Using clinical SARS CoV-2 swab samples, it could be shown that the use of the buffer B followed by direct PCR without RNA extraction is clearly superior to direct PCR from a sample without extraction. This shows that using buffer B sufficient sensitivity can be reached without the need for previous RNA extraction (Tables 10 and 11 ).

Further, the results as shown in Figure 9 show that using the SARS CoV-2 virus culture in buffer B directly in the PCR, the ct values were on average about 2 better than using conventional RNA extraction prior to the PCR.

Summary

A sample treated with buffer B can be used directly in the PCR without RNA extraction. Buffer B thus offers an advantage over other virus media suited for cell lysis and transport, such as eNat virus transport medium, which contain guanidinium thiocyanate inhibiting PCR reactions.

Classical PCR diagnostics are generally performed in fully equipped laboratories, not in the field or point-of-care settings. Accordingly, these laboratories generally have the appropriate equipment for RNA extraction. A sample lysed and stored in buffer B can go through the RNA extraction quite normally and be examined in a PCR without loss of sensitivity compared to a sample stored in other virus transport media.

In laboratory-based standard diagnostics, the advantage of the buffer B, i.e. a buffer according to the invention, is the possibility of performing a PCR diagnosis without RNA extraction, while still allowing the possibility to go through the normal sample processing route including RNA extraction without compatibility problems.

EXAMPLE 3

LAMP

Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection (Notomi, T, Okayama, H, Masabuchi, H, Yonekawa, T, Watanabe, K, Amino, N, Hase, T Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000 Jun 15; 28(12): e63) that, due to its isothermal functionality and options for visual evaluation, only minor technological requirements are necessary and is therefore ideal for use in low-resource regions or potentially as a point-of-care method. Various commercial LAMP systems are available for the detection of RNA viruses, such as SARS CoV-2, whereby the sample analysis is usually performed with prior RNA extraction.

The compatibility of a buffer according to the invention with two commercial LAMP systems was investigated. In particular, the efficiency of SARS CoV-2 detection on nonextracted sample material with or without treatment with buffer B was compared.

LAMP system “Eazyplex SARS CoV-2”, Amplex Diagnostics GmbH

The Eazyplex system from Amplex Diagnostics (Cat. no. 7680) contains lyophilized reagents that were used in combination with the proprietary lysis buffer "RALF". A sample was added directly to the RALF buffer without RNA extraction and the lyophilizate of the reagents was dissolved with this suspension. The LAMP reaction was carried out in the proprietary isothermal amplification device "Genie" with the associated software (Version 2.34.5, Amplex Diagnostics). The detection takes place via an intercalating fluorescent dye. Two target genes, N and ORF8, are detected for SARS CoV-2 detection. A sample was evaluated as SARS CoV-2 positive if at least one of the two target genes was positive with valid controls at the same time.

A panel of 64 PCR-confirmed SARS CoV-2 positive nose I throat swab specimens in PBS was used. The samples had ct values between 19-40 in the PCR (PCR E-Gen, Corman et al. 2020, PCR System MIC, PCR reagents Qiagen OneStep).

1 . Implementation according to the manufacturer's protocol

25 pl sample was mixed with 500 pl RALF buffer. Of this, 25 pl each were used for the LAMP reaction. Results are shown in Table 12 and Figure 5b.

2. Implementation with buffer B

25 pl sample were mixed with 5 pl Buffer B/ RNase inhibitor (= 0.5 pl RNase inhibitor [40U/pl] in 100 pl 6-fold buffer B) and incubated for 5 min at room temperature. Buffer B’s final concentration was 1 -fold. 500 pl of RALF buffer were added and 25 pl of each was used for the LAMP reaction. Results are shown in Table 13 and Figure 5a.

Results

Table 12: Results using manufacturer’s protocol

Table 13: Results using buffer B

Summary

The use of samples in buffer B is fully compatible with the Eazyplex-SARS-CoV-2 LAMP system and leads to improved sensitivity and shorter detection times.

LAMP system "Loopamp SARS CoV-2", Eiken Chemical Co. Ltd., Japan

The Loopamp System from Eiken Chem (LMC403) contains lyophilized reagents and a liquid SARS CoV-2-specific primer mix. The primer mix as provided by the manufacturer and sample were mixed and the lyophilizate of the reagents was dissolved and the LAMP reaction was started. The detection is based on calcein and was carried out either in a turbidimeter or in a qPCR device in the FAM channel.

The manufacturer recommends the use of extracted RNA for SARS CoV-2 detection with the Loopamp system. It was tested whether with the use of the buffer the time-consuming and cost-intensive and mostly device-dependent RNA extraction step can be omitted.

A panel of 65 PCR-confirmed SARS CoV-2 positive nose I throat swab specimens in PBS was used. The samples had ct values between 19 to 40 in the PCR (PCR E-Gen, Corman et Al. 2020, PCR System MIC, PCR reagents Qiagen OneStep (Cat. No 210212)).

The sample panel was examined in four different experiments with the Loopamp system. Particulars are given below. The reactions were carried out in a qPCR device (MIC, bms) (60 cycles, 62.5°C., 40 sec, 1 cycle 95°C., 120 sec) and measured in the FAM channel. 1 . without RNA extraction

Procedure: 10 l sample + 15 pl primer mix as provided by the manufacturer to dissolve the lyophilizate and start the reaction

2. with Loopamp Viral RNA Extraction kit

Procedure: 10 pl sample + 400 pl Loopamp extraction buffer; from this mixture 10 pl of + 15 pl primer mix as provided by the manufacturer to dissolve the lyophilizate and start the reaction.

3. with buffer B

Procedure: 10 pl sample + 2 pl buffer B (6-fold) I RNase inhibitor + 15 pl primer mix as provided by the manufacturer to dissolve the lyophilizate and start the reaction

4. RNA extracted from Qiagen Viral RNA Kit

Procedure: 10 pl solution of extracted RNA + 15 pl primer mix as provided by the manufacturer to dissolve the lyophilizate and start the reaction.

Results

Table 14: Summary of the results with the Loopamp system ct<25 ct<30 ct<35 ct<40

* 33 samples excluded, due to incompletely dissolved lyophilsate “No” refers to “Number of samples” The treatment of the samples with buffer leads to a significant improvement in the results compared to non-extracted, untreated samples with sensitivity closer to the extracted samples, showing sufficient sensitivity (Table 14 and Figure 6).

Summary

The use of samples in buffer B is compatible with the Loopamp system and leads to improved sensitivity and shorter detection times compared to non-extracted samples in PBS.

EXAMPLE 4

Rapid antigen tests

In addition to the gold standard of RT-PCR, rapid antigen tests are another cornerstone of virus diagnostics. A positive result in the rapid tests designed for point-of-care use, nowadays is usually confirmed with a PCR test. In order to avoid a second sampling for the confirmatory test, which is unpleasant for the patient and not always possible for logistical reasons, a buffer system that is suitable for both rapid tests and PCR would be advantageous. The frequently used eNat virus transport medium is not compatible with rapid antigen tests, and the UTM, which is also popular, is only partially compatible.

Method

Beta-propiolactone-inactivated SARS COV-2 virus material, which was diluted in buffer B, was used for the test. Two virus concentration levels were tested in duplicates (10⁵ PFU/ml and 10³ PFU/ml). The higher concentration is generally easy to detect in rapid antigen tests; at the lower concentration, some rapid tests already reach their detection limit.

Two variants were tested:

Variant 1 : The sample diluted in buffer B was applied directly to the rapid test cassette Variant 2: The sample diluted in buffer B was mixed with the manufacturer's rapid test buffer according to the manufacturer's instructions and then applied to the cassette.

As controls the two virus concentration levels where either applied in PBS instead of buffer B or in manufacturer's buffer for the rapid test.

The tests were evaluated after the time specified by the manufacturer. The intensity of the visible lines was assessed using a comparative color scale with a value between 0 (no lines visible) and 9 (very strong line).

A total of 7 different rapid tests based on 3 different detection mechanisms (gold particles, latex particles, fluorescence) were tested (Roche SARS CoV-2 Rapid Antigen Test (Cat. no. 9901 -NCOV-01 G), Abbott Panbio CoV-19 Ag Test (Cat. no. 41 FK10), Healgen Coronavirus Rapid Test Cassette (Swab) (Cat. no. GCCOV-502a), Nal von Minden NADAL Covid-19 Ag Test (Cat. no. 243103N-20), Schebo SARS-CoV-2 Ag Rapid Test Kit (Fluorescence Immunochromatography) (Cat. no. 34), Quidel SOFIA SARS Antigen FIA (Cat. no. 20374), Biomerica COVID-19 Antigenschnelltest (Nasopharyngeal- Abstrich), Cat. no. 1509A.)

Further, the compatibility of the buffer B with SARS CoV-2 antigen rapid tests in the lateral flow format was tested for the Roche Covid-19 AG rapid test (Cat. No. 9901 -NCOV-01 G) using either the running buffer contained in the kit (running buffer) or buffer B. The time until appearance of the results was measured. To this end, 50 pl of a SARS CoV-2 positive sample (nasal I throat swab dissolved in PBS) was mixed with 300 pl buffer (running buffer or buffer B) and 75 pl of this was applied to the test cassette. The evaluation took place after 15 minutes. Results are presented in Table 16, below. Results

Table 15. Results of the rapid tests. The line intensity is indicated on a scale from 0 - 9.

Overall, the higher virus concentration could be reliably detected with strong line intensity using buffer B in both variant 1 and variant 2 for all seven manufacturers tested.

Surprisingly, buffer B showed good results in terms of sensitivity for most of the tests applied and was by far better than the control (see Table 15 and Figure 8).

Table 16. Results of the rapid tests comparing buffer B and the running buffer as supplied with the Roche kit. The time frame until line appearance is indicated

Summary

Overall, the buffer B (according to the invention) (either used in variant 1 or 2) leads to better results. The buffer according to the invention is therefore compatible with SARS CoV-2 antigen lateral flow rapid tests.

Without being bound by theory, it is thought that this is due to the effective lysis of the SARS CoV-2 virus particles by buffer B, which releases virus proteins, in particular the nucleocapsid protein located inside the virus. Almost all SARS CoV-2 rapid antigen tests currently available on the market (as well as all those tested here) detect the nucleocapsid protein.

The results prove that the buffer according to the invention can in addition to DNA or RNA detection methods also be used in methods to detect specific (virus) proteins and thereby providing for a universal buffer that makes purification steps superfluous.

Using buffer B, the control line in the Roche rapid test appeared a little earlier; there were further differences when developing the test line. In 2 of 6 tested samples the test cassette with buffer B showed a better line intensity. The use of buffer B performs better than the buffer supplied with the test as regards the contrast of the lines.

EXAMPLE 5

Sequencing

In the course of the emergence of new viruses und variants, such as during SARS CoV- 2 pandemic, epidemiological investigations of individual outbreak clusters, genome sequencing is becoming increasingly important. A buffer system that preserves the virus RNA in a quality adequate for whole genome sequencing would be advantageous. To test whether the RNA is fragmented by the buffer according to the invention (which would lead to a poorer quality of the sequencing results) or whether it remains intact, the following example was conducted.

Method

A solution of 950 pl 1x buffer B was mixed with 50 pl native SARS CoV-2 virus material in a concentration of 2 x 10⁵ PFU I ml (strain IMB muc-1 , EPI-ISL-406862, corresponds to wild type). After an incubation of approx. 2 h, a magnetic beads-based RNA extraction was carried out and the sample was sequenced using Illumina MiSeq.

Results

There was no loss of quality in the sequence data compared to other diagnostic samples stored in other transport media, such as VTM (viral transport media; BioServlIK, Cat No: BSV-VTM-001 ) or eNat (Cat No. Copan 80608C).

Summary

The use of the buffer according to the invention for sample transport is compatible with further sample processing for whole genome sequencing.

Disclosed sequences

* Used Probe contained dye and quencher at the 5’-end (FAM) and at the 3’-end (Black Hole Quencer (BHQ), “BHQ1”), respectively

** Probe may also contain dye and/or quencher as needed, e.g. 5’-end (FAM) and at the 3’-end (BHQ1), respectively.

ITEMS OF THE INVENTION.

The invention in particular relates to the following items:

1. A method to inactivate an RNA or DNA virus comprising contacting at least one sample with a lysis buffer to produce a lysate, wherein the lysis buffer comprises a non-ionic surfactant, glycerol, and at least one salt.

2. The method according to item 1 , wherein the lysis buffer does not contain a guanidinium salt.

3. The method according to items 1 or 2, wherein the at least one non-ionic surfactant has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group.

4. The method according to any one of items 1 to 3, wherein the at least one non-ionic surfactant is Triton X-100.

5. The method according to any one of items 4, wherein the lysis buffer comprises Triton X-100 in an amount from about 0.05% to about 20%, preferably 0.3% to 3.5%, more preferably 0.5% to 3%.

6. The method according to any one of items 1 to 5, wherein the salt is disodium phosphate.

7. The method according to any one of items 6, wherein the lysis buffer comprises disodium phosphate in a concentration from about 1 mM to about 50 mM, preferably 3 mM to 30 mM, more preferably 4 mM to 25 mM

8. The method according to any one of items 1 to 7, wherein the lysis buffer further comprises one or more of the following: Tris-HCI, dithiothreitol (DTT), RNase-free water, RNase inhibitor, and mixtures thereof.

9. The method according to any one of items 1 to 8, wherein the pH of the lysis buffer is from about 7.5 to about 8.5.

10. The method according to any one of items 1 to 9, wherein the lysis buffer comprises:

- 64 - 3% Triton-X 100

10% Glycerol

2 mM DTT

25 mM Na₂HPO₄

25 mM Tris HCI;

RNAse-free water; and has a pH of 7.8; and wherein the lysis buffer does not contain a guanidinium salt. The method according to any one of items 1 to 9, wherein the lysis buffer comprises:

0.5% Triton-X 100

1.67% glycerol

0,33 mM DTT

4,17 mM Na₂HPO₄

4,17 mM Tris HCI;

RNAse-free water; and has a pH of 7.8; and wherein the lysis buffer does not contain a guanidinium salt. The method according to any one of items 1 to 11 , wherein the sample is a biological sample. The method according to item 12, wherein the biological sample is collected using a swab. The method according to item 12 or 13, wherein the biological sample is a nasal swab. The method according to any one of items 1 to 14, wherein contacting the at least one sample with the lysis buffer comprises incubating the mixture of the lysis buffer and the sample for 5 minutes at room temperature to produce a lysate. The method according to any one of items 1 to 19, further comprising centrifuging the lysate and collecting the supernatant.

- 65 - A method preparing a sample intended for the analysis for the presence of an RNA or DNA virus comprising inactivating the virus in at least one sample by the method according to any one of items 1 to 17 to produce a lysate and thereby inactivating the virus, and storing and/or transporting the lysate. The method according to item 18, wherein storing and/or transporting is conducted at a temperature between -80°C to 45°C, preferably -20°C to room temperature, more preferably 4°C to room temperature. The method according to item 18 or 19, wherein storing and/or transporting is performed for 12 hours to 1 week, preferably 1 to 5 days. A method for detecting a virus in a sample comprising preparing the sample with the method according to any one of items 18 to 20; analyzing the lysate for the presence of a virus in the sample by detecting virus DNA, virus RNA or a protein antigen in the lysate. The method according to item 21 , wherein the method does not comprise an RNA, DNA and/or protein extraction step. The method according to item 21 or 22, wherein analyzing the lysate is performed by detecting or sequencing virus specific DNA, RNA and/or antigen. The method according to any one of items 21 to 23, wherein said analyzing comprises the amplification of DNA or RNA from said lysate. The method according to item 24, wherein said amplifying of at least one nucleic acid in the lysate is performed by PCR, qPCR, RT-PCR, RT-qPCR, or loop-mediated isothermal amplification (“LAMP”). The method according to any one of items 21 to 25, wherein, if the virus is an RNA virus, The method according to any one of items 24 to 26, wherein the amplifying of at least one nucleic acid in the lysate is performed by one-step RT-qPCR.

- 66 - A method according to any one of items 1 to 27, wherein the virus is an enveloped virus, preferably an enveloped RNA virus, more preferably a virus of the family Coronaviridae. The method according to item 28, wherein detection of virus RNA is performed by first reverse transcribing RNA to obtain cDNA and amplifying the cDNA or a part thereof by using a set of primers derived from the RNA of the virus, virus is a coronavirus. The method according to item 28 or 29, wherein the coronavirus is SARS CoV-2. The method according to item 28 or 29, wherein the coronavirus is a variant of SARS CoV-2. The method according to any one of items 29 to 31 , wherein the set of primers is directed to a viral RNA gene selected from the group consisting of an E gene, an N gene, and an RdRP gene. The method according to item 32, wherein the set of primers is directed to the E gene and comprise or consist of SEQ ID NO: 2 and/or SEQ ID NO: 3, or a complement thereof. The method according to item 32, wherein the set of primers is directed to the RdRP gene and comprise or consist of SEQ ID NO: 5 and/or SEQ ID NO: 6, or a complement thereof. The method according to any one of item 21 to 23, wherein analyzing the lysate for the presence of a virus is performed by detecting the presence of a virus protein or a virus antigen. The method according to item 35, wherein presence of a virus protein or a virus antigen is performed by an immunological assay, preferably by a lateral flow assay. The method according to any one of items 21 to 36, wherein the step of analyzing the lysate for the presence of a virus in the sample comprises the detection of virus DNA or virus RNA, and virus protein or antigen in two aliquots of the lysate.

- 67 - A kit comprising a lysis buffer, and two or more vessels for aliquotation of a lysate; wherein the lysis buffer comprises at least one non-ionic surfactant, glycerol, and at least one salt. The kit according to item 38, further comprising at least one reagent for amplification of a target nucleic acid. The kit according to item 38 or 39 comprising at least one primer and/or at least one probe for amplification of a target nucleic acid. The kit according to any one of items 38 to 40, wherein the kit is a kit for detection of RNA using one-step RT-qPCR. The kit according to item 41 , comprising one or more of at least one reverse transcriptase, at least one DNA polymerase, an RNase inhibitor, nucleotides, primers, probes, labels, or any combination thereof. The kit according to any one of items 38 to 42, wherein the target nucleic acid is derived from a RNA virus. Use of the lysis buffer lysis buffer comprising 1 to 5% Triton X-100 and one or more of the following components: 5% to 20% glycerol, 0.5 mM to 4 mM DTT, 10-50 mM Na2HPO4, and 10 mM to 50 mM Tris-HCI in a method for inactivating, transporting and/or storing, and analyzing a sample for the presence of a virus. Use of the lysis buffer according to item 44, wherein the lysis buffer does not contain a guanidinium salt. Use of the lysis buffer according to item 44 or 45, wherein the lysis buffer contains about 3% Triton X-100 and one or more of the following components: about 10% glycerol, about 2 mM DTT, about 25 mM Na2HPO4, and about 25 mM Tris-HCI. Use of the lysis buffer according to any one of items 44 to 46, wherein the pH of the lysis buffer is from about 7.5 to about 8.0. Use of the lysis buffer according to any one of items 44 to 47, further comprising RNase-free water.

- 68 - Use of the lysis buffer according to item 48, wherein the RNase-free water is included in an amount from about 1 :15 to 1 :1 (lysis buffer: RNase-free water). Use of the lysis buffer according to any one of items 44 to 49, further comprising an RNAse inhibitor.

- 69 -

Claims

1 . A method for detecting a virus in a sample comprising inactivating the virus by contacting said sample with a lysis buffer according to the invention to produce a lysate; taking one or more aliquot of the lysate; analyzing the one or more aliquot for the presence of a virus in the sample by detecting an antigen of the virus in the lysate; optionally storing and/or transporting the remainder of the lysate and/or a further aliquot thereof; and and analyzing the remainder of the lysate or the further aliquot thereof for the presence of a virus in the sample by detecting virus DNA or virus RNA in the lysate or the further aliquot thereof; wherein the lysis buffer comprises a non-ionic surfactant, glycerol, and at least one salt.

2. The method according to claim 1 , wherein the lysis buffer does not contain a guanidinium salt.

3. The method according to claim 1 or 2, wherein the at least one non-ionic surfactant has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group.

4. The method according to any one of claims 1 to 3, wherein the at least one non-ionic surfactant is Triton X-100.

5. The method according to any one of claims 4, wherein the lysis buffer comprises Triton X-100 in an amount from about 0.05% to about 20%, preferably 0.3% to 3.5%, more preferably 0.5% to 3%.

6. The method according to any one of claims 1 to 5, wherein the salt is disodium phosphate.

- 70 - The method according to any one of claims 6, wherein the lysis buffer comprises disodium phosphate in a concentration from about 1 mM to about 50 mM, preferably 3 mM to 30 mM, more preferably 4 mM to 25 mM The method according to any one of claims 1 to 7, wherein the lysis buffer further comprises one or more of the following: Tris-HCI, dithiothreitol (DTT), RNase-free water, RNase inhibitor, and mixtures thereof. The method according to any one of claims 1 to 8, wherein the pH of the lysis buffer is from about 7.5 to about 8.5. The method according to any one of claims 1 to 9, wherein the lysis buffer comprises:

3% Triton-X 100

10% glycerol

2 mM DTT

25 mM Na₂HPO₄

25 mM Tris HCI;

RNAse-free water; and has a pH of 7.8; and wherein the lysis buffer does not contain a guanidinium salt. The method according to any one of claims 1 to 9, wherein the lysis buffer comprises:

0.5% Triton-X 100

1.67% glycerol

0.33 mM DTT

4.17 mM Na₂HPO₄

4.17 mM Tris HCI;

RNAse-free water; and has a pH of 7.8; and wherein the lysis buffer does not contain a guanidinium salt.

- 71 - The method according to any one of claims 1 to 11 , wherein the sample is a biological sample. The method according to claim 12, wherein the biological sample is collected using a swab. The method according to claim 12 or 13, wherein the biological sample is a nasal swab. The method according to any one of claims 1 to 14, wherein the lysate and the one or more aliquots are directly used in the steps of analyzing for the presence of a virus by detecting virus DNA or virus RNA, and the virus antigen.

- 72 -