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WO2024031508A1 - Method for detecting multiple target nucleic acids - Google Patents

Method for detecting multiple target nucleic acids Download PDF

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Publication number
WO2024031508A1
WO2024031508A1 PCT/CN2022/111673 CN2022111673W WO2024031508A1 WO 2024031508 A1 WO2024031508 A1 WO 2024031508A1 CN 2022111673 W CN2022111673 W CN 2022111673W WO 2024031508 A1 WO2024031508 A1 WO 2024031508A1
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Prior art keywords
digital magnetic
group
nucleic acid
magnetic beads
probe
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PCT/CN2022/111673
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French (fr)
Chinese (zh)
Inventor
王其伟
黄硕
余进文
耿春雨
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武汉华大智造科技有限公司
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Priority to PCT/CN2022/111673 priority Critical patent/WO2024031508A1/en
Publication of WO2024031508A1 publication Critical patent/WO2024031508A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention provides a multi-target nucleic acid detection method, specifically involving a method of using digital magnetic beads to achieve rapid detection and simultaneous typing of multiple pathogenic microorganisms.
  • HPV Human papillomavirus
  • HPV Human papillomavirus
  • Papovaviridae a spherical DNA virus that can cause the proliferation of squamous epithelium in human skin and mucous membranes.
  • HPV subtypes known today, and dozens of them have been proven to cause uncomfortable symptoms in humans, such as common warts and genital warts (genital warts).
  • High-risk HPV can even cause a variety of malignant tumors such as cervical cancer, anal cancer, vaginal cancer, mouth and nose cancer, vulvar cancer, and penile cancer, among which cervical cancer is the most common.
  • HPV screening can effectively reduce the incidence and mortality of cervical cancer.
  • the experience of countries or regions with complete screening systems such as North America, Australia and Europe has proven that HPV screening can effectively reduce the incidence and mortality of cervical cancer, and large-scale cervical cancer screening is important for improving the quality of human life. Promoting effect.
  • HPV typing is mostly achieved by real-time fluorescence quantitative PCR (qPCR).
  • qPCR real-time fluorescence quantitative PCR
  • a fluorescent group coupled to a probe is added to the reaction system, and the entire PCR process is monitored in real time using fluorescence signal accumulation.
  • the unknown template is quantitatively analyzed through a standard curve.
  • Taqman probe labeled with fluorescein is mixed with the template DNA, a thermal cycle of high-temperature denaturation, low-temperature renaturation, and appropriate temperature extension is completed, and the rules of the polymerase chain reaction are followed.
  • the Taqman probe complementary to the template DNA is cut off, and the fluorescence
  • the protein is free in the reaction system and emits fluorescence under specific light excitation; as the number of cycles increases, the amplified target gene fragment grows exponentially, and through real-time detection of the corresponding changes in fluorescence signal intensity with amplification, Calculate the Ct value and use several standards with known template concentrations as controls to determine the copy number of the target gene in the sample to be tested.
  • the HPV subtypes that can be distinguished simultaneously using this scheme are limited.
  • the invention provides a multi-target nucleic acid detection method, specifically involving a method of using digital magnetic beads to achieve rapid detection and simultaneous typing of multiple pathogenic microorganisms.
  • the invention provides a method for detecting n target nucleic acids, which includes the following steps:
  • the nucleic acid to be tested is subjected to an amplification reaction; the purpose of the amplification reaction is: when the nucleic acid to be tested contains a target nucleic acid, an amplification product with a terminal modified with group A is obtained through the amplification reaction;
  • each digital magnetic bead coupled to the probe can bind to one target nucleic acid molecule; the digital magnetic beads coupled to the probe Beads are obtained by coupling a probe used to capture target nucleic acids and digital magnetic beads; each digital magnetic bead coupled to a probe is obtained by coupling a probe used to capture target nucleic acids and a digital magnetic bead. Obtained; each type of digital magnetic beads has the same barcode, and different types of digital magnetic beads have different barcodes;
  • the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
  • the reaction system is tested as follows: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains the target nucleic acid and/or determine whether the nucleic acid to be tested contains n kinds of target nucleic acids based on the barcode and light signal. Which type or types;
  • n is a natural number above 2.
  • the amplification reaction is an asymmetric PCR amplification reaction.
  • the purpose of the asymmetric PCR amplification reaction is: when the nucleic acid to be tested contains the target nucleic acid, a single-stranded amplification product with a group A modified at the end is obtained through the amplification reaction.
  • the concentrations of the upstream primer and the downstream primer in the amplification system are different.
  • the molar ratio of the upstream primer and the downstream primer in the amplification system can be 0.1:10.
  • the primer pair used in the amplification reaction consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with the group A.
  • the group A and the group B are affinity groups for each other.
  • the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
  • the group B is streptavidin.
  • the light signal is a fluorescence signal.
  • the group C is phycoerythrin.
  • the detection element is streptavidin phycoerythrin (SA-PE).
  • the 5' end of the probe is modified with NH 2 C 12 .
  • n 2
  • the method provided by the invention can be used for joint detection of multiple pathogenic microorganisms.
  • the various pathogenic microorganisms include but are not limited to the following: influenza virus, new coronavirus, poxvirus, adenovirus, etc., or a variety of pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Streptococcus, etc.
  • the method provided by the invention can be used for joint detection of multiple biomarkers.
  • the various biomarkers include, but are not limited to, the following: nucleic acids characteristic of liver cancer markers (such as alpha-fetoprotein) in peripheral blood, nucleic acids characteristic of colorectal cancer markers in peripheral blood, etc.
  • the invention also provides a kit for detecting n kinds of target nucleic acids, including an amplification primer set composed of n primer pairs, a digital magnetic bead set composed of n kinds of digital magnetic beads coupled to probes, and a detection kit. element;
  • each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
  • the digital magnetic beads for coupling probes are obtained by coupling the probe used to capture the target nucleic acid with the digital magnetic beads; each type of digital magnetic beads for coupling probes is obtained by coupling one type of digital magnetic beads for capturing target nucleic acids.
  • the target nucleic acid probe is coupled to a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcodes;
  • the detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
  • n is a natural number above 2.
  • the molar ratio of the upstream primer and the downstream primer can be 0.1:10.
  • the group A and the group B are affinity groups for each other.
  • the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
  • the group B is streptavidin.
  • the light signal is a fluorescence signal.
  • the group C is phycoerythrin.
  • the detection element is streptavidin phycoerythrin (SA-PE).
  • the 5' end of the probe is modified with NH 2 C 12 .
  • n 2
  • the kit provided by the invention can be used for joint detection of multiple pathogenic microorganisms.
  • the various pathogenic microorganisms include but are not limited to the following: influenza virus, new coronavirus, poxvirus, adenovirus, etc., or a variety of pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Streptococcus, etc.
  • the kit provided by the invention can be used for joint detection of multiple biomarkers.
  • the various biomarkers include, but are not limited to, the following: nucleic acids characteristic of liver cancer markers (such as alpha-fetoprotein) in peripheral blood, nucleic acids characteristic of colorectal cancer markers in peripheral blood, etc.
  • the present invention also provides a method for detecting HPV, which includes the following steps:
  • the amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV ;Each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
  • the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
  • the reaction system is subjected to the following detection: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains HPV and/or determine which of the n types of HPV is contained in the nucleic acid to be tested based on the barcode and light signal.
  • detect the barcode and light signal of the magnetic beads determine whether the nucleic acid to be tested contains HPV and/or determine which of the n types of HPV is contained in the nucleic acid to be tested based on the barcode and light signal.
  • detect the barcode and light signal of the magnetic beads determine whether the nucleic acid to be tested contains HPV and/or determine which of the n types of HPV is contained in the nucleic acid to be tested based on the barcode and light signal.
  • n is a natural number above 2.
  • the PCR amplification is asymmetric PCR amplification.
  • the concentrations of the upstream primer and the downstream primer in the amplification system are different.
  • the molar ratio of the upstream primer and the downstream primer in the amplification system can be 0.1:10.
  • reaction system of the asymmetric PCR amplification is shown in Table 1.
  • the group A and the group B are affinity groups for each other.
  • the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
  • the group B is streptavidin.
  • the light signal is a fluorescence signal.
  • the group C is phycoerythrin.
  • the detection element is streptavidin phycoerythrin (SA-PE).
  • the 5' end of the probe is modified with NH 2 C 12 .
  • n 2
  • the amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV16 Amplify the characteristic nucleic acid of HPV18, consisting of HPV18-F and HPV18-R.
  • the digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is a probe that will be used to capture the characteristic nucleic acid of HPV16.
  • the probe coupled to a digital magnetic bead for capturing the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupled to the probe is used to capture the characteristic nucleic acid of HPV18.
  • the probe is coupled to a digital magnetic bead and is used to capture the characteristic nucleic acid of HPV18, named HPV18-P.
  • the nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleoside of HPV16-P
  • the acid sequence is shown in SEQ ID NO: 3; the nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleic acid sequence of HPV18-P is shown in SEQ ID NO: 4.
  • the nucleotide sequence is shown in SEQ ID NO: 6.
  • the invention also provides a kit for detecting HPV, including an amplification primer set, a digital magnetic bead set and a detection element;
  • the amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV; each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with Group A;
  • the digital magnetic bead set consists of n types of digital magnetic beads coupled to probes; the digital magnetic beads coupled to probes are obtained by coupling the probe used to capture the target nucleic acid and the digital magnetic beads; each coupled probe The digital magnetic beads are obtained by coupling a probe used to capture the characteristic nucleic acid of a type of HPV with a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcode;
  • the detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
  • n is a natural number above 2.
  • the molar ratio of the upstream primer and the downstream primer can be 0.1:10.
  • the group A and the group B are affinity groups for each other.
  • the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
  • the group B is streptavidin.
  • the light signal is a fluorescence signal.
  • the group C is phycoerythrin.
  • the detection element is streptavidin phycoerythrin (SA-PE).
  • the 5' end of the probe is modified with NH 2 C 12 .
  • n 2
  • the amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV16 Amplify the characteristic nucleic acid of HPV18, consisting of HPV18-F and HPV18-R.
  • the digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is a probe that will be used to capture the characteristic nucleic acid of HPV16.
  • the probe coupled to a digital magnetic bead for capturing the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupled to the probe is used to capture the characteristic nucleic acid of HPV18.
  • the probe is coupled to a digital magnetic bead and is used to capture the characteristic nucleic acid of HPV18, named HPV18-P.
  • the nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleoside of HPV16-P
  • the acid sequence is shown in SEQ ID NO: 3; the nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleic acid sequence of HPV18-P is shown in SEQ ID NO: 4.
  • the nucleotide sequence is shown in SEQ ID NO: 6.
  • the preparation method of digital magnetic beads coupled to probes is specifically as described in step 2 of Example 2.
  • the method for reacting the hybridization product with the detection element is specifically as described in steps 1 to 6 of step three of embodiment 2.
  • the method for reacting the hybridization product with the detection element is specifically as described in Steps 1 to 8 of Step 3 of Embodiment 2.
  • the streptavidin phycoerythrin shown can specifically be: Abcam, product number ab239759.
  • the digital magnetic beads may be digital magnetic beads produced by Taiwan Wen Yin.
  • the existing technical solution cannot achieve joint detection of targets, resulting in high costs and long total operation time.
  • the present invention realizes the joint detection of HPV16 and HPV18.
  • the solution of the present invention can be extended to joint detection of multiple targets. For example, joint testing of more than a dozen or even hundreds of subtypes of HPV.
  • the multiple encoding properties of the digital magnetic beads in the present invention enable joint detection of multiple targets. This feature enables single-hole detection of multiple nucleic acid targets in a single sample (that is, detection of multiple HPV subtypes). Single-well simultaneous detection) can improve detection efficiency, greatly reduce workload and reduce reagent costs; 2 In the DNA target amplification process, an asymmetric PCR scheme is used to make the DNA to be tested directly single-stranded DNA, thereby omitting The high-temperature denaturation and low-temperature incubation processes are eliminated, which simplifies the experimental steps and avoids the problem of cross-contamination of samples due to aerosols generated by high temperatures.
  • Figure 1 shows the general process for nucleic acid detection using digital magnetic beads.
  • Figure 2 is a joint inspection flow chart.
  • Figure 3 shows the signal-to-noise ratio of HPV16 and HPV18 obtained by joint detection of HPV16 and HPV18 using this joint detection technology.
  • test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified. Unless otherwise specified, the quantitative tests in the examples were repeated at least three times, and the results were averaged.
  • the direction of DNA molecules in the examples is 5' ⁇ 3' direction.
  • the primers and probes used to detect HPV16 are as follows:
  • HPV16-F upstream primer: GCACAGGGCCACAATAATGG;
  • HPV16-R downstream primer: TripleBiotin-CATAACGTCTGCAGTTAAGGTTATT;
  • HPV16-P (probe): NH 2 C 12 -TGTGCTGCCATATCTACTTCAGA.
  • the primers and probes used to detect HPV18 are as follows:
  • HPV18-F upstream primer: GCACAGGGTCATAACAATGG;
  • HPV18-R downstream primer: TripleBiotin-CATGTTCTGCTATACTGCTTAAAT;
  • HPV18-P (probe): NH 2 C 12 -AGTCTCCTGTACCTGGGCAA.
  • the above primer probes are all single-stranded DNA molecules, and the direction is 5' ⁇ 3'.
  • the 5' end is modified with TripleBiotin (also known as Triple-Biotin).
  • the 5' end is modified with NH 2 C 12 .
  • the nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1.
  • the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2.
  • the nucleotide sequence of HPV16-P is shown in SEQ ID NO: 3.
  • the nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4.
  • the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5.
  • the nucleotide sequence of HPV18-P is shown in SEQ ID NO: 6.
  • the downstream primers are HPV16-R and HPV18-R.
  • the upstream primers are HPV16-F and HPV18-F.
  • KAPA2G Fast Multiplex Mix American KAPA Biosystems company, product catalog number is KK5802.
  • step 2 After completing step 1, centrifuge briefly and collect the liquid at the bottom of the PCR tube.
  • Digital magnetic beads Digital magnetic beads are purchased from Winmems, Taiwan, website:
  • Digital magnetic beads are etched with different barcodes that can be identified by white light; when magnetic beads with different numbers are mixed together, they can be identified and classified through the barcodes. Digital magnetic beads are stored at 4°C. Digital magnetic beads are composed of a matrix and magnetic material (the matrix is polystyrene and the magnetic material is ferroferric oxide). The outer surface is modified with carboxyl groups and the outer surface is etched with different barcodes that can be recognized by white light using photolithography. Magnetic beads The shape is rectangular and its specification is 25*35mm.
  • each probe is coupled to digital magnetic beads with a specific barcode, so that each probe has a specific barcode.
  • the probe is HPV16-P or HPV18-P in Example 1.
  • HPV16-P specifically recognizes HPV16.
  • HPV18-P specifically recognizes HPV18-P.
  • Each probe is coupled to a digital magnetic bead (a digital magnetic bead refers to a digital magnetic bead with the same barcode), and the following steps are performed:
  • step 2 After completing step 1, add 500 ⁇ L isopropyl alcohol to the EP tube and vortex for 30 seconds to mix thoroughly.
  • step 3 centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
  • step 3 place the EP tube on a magnetic stand for 2 minutes to allow all digital magnetic beads to precipitate.
  • step 4 After completing step 4, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
  • step 5 After completing step 5, add 500 ⁇ L of washing buffer 1 to the EP tube and vortex for 30 seconds to mix thoroughly.
  • Washing buffer 1 contains 0.1M 2-(N-morpholine)ethanesulfonic acid (MES) and 0.05% Tween 20 (Tween 20), and the balance is water.
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Tween 20 0.05% Tween 20
  • step 6 centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
  • step 7 After completing step 7, keep the EP tube on the magnetic stand for 2 minutes to allow all digital magnetic beads to settle.
  • step 8 After completing step 8, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
  • step 11 After completing step 10, add 500 ⁇ L of washing buffer 2 to the EP tube and vortex for 30 seconds to mix thoroughly.
  • Washing buffer 2 (pH 5.0): contains 0.1M 2-(N-morpholine)ethanesulfonic acid (MES), and the balance is water.
  • MES 2-(N-morpholine)ethanesulfonic acid
  • step 12 centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
  • step 13 After completing step 12, place the EP tube on a magnetic stand for 2 minutes to allow all digital magnetic beads to precipitate.
  • step 13 After completing step 13, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
  • step 14 After completing step 14, add 28 ⁇ L cleaning buffer 2, 15 ⁇ L EDC solution, 3 ⁇ L NHS solution and 4 ⁇ L 100mM probe solution to the EP tube, and mix thoroughly.
  • EDC solution Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in washing buffer 2 to make the concentration of EDC 20 mg/mL. Ready for use.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Preparation method of NHS solution Dissolve N-hydroxysuccinimide (NHS) in washing buffer 2 to make the concentration of NHS 20 mg/mL. Ready for use.
  • NHS N-hydroxysuccinimide
  • step 16 place the EP tube in a mixer and react at 1400 rpm at room temperature for 30 minutes in the dark.
  • step 16 wash the magnetic beads once with 0.2g/L Tween-20 aqueous solution, and discard the supernatant.
  • step 17 wash the magnetic beads once with 1g/L sodium dodecyl sulfate (SDS) aqueous solution, and discard the supernatant.
  • SDS sodium dodecyl sulfate
  • step 18 After completing step 18, add 100 ⁇ L TE buffer, which is the digital magnetic bead solution of the coupled probe, into the EP tube and store at 4°C.
  • the digital magnetic bead solution coupled to HPV16-P and the digital magnetic bead solution coupled to HPV18-P were obtained respectively.
  • Hybridization buffer (pH8.0): Contains 0.15M tetramethylammonium chloride (TMAC), 75mM tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 6mM ethylenediaminetetraacetic acid (EDTA), 1.5g /L sodium lauryl sarcosinate (Sarkosyl), the balance is water.
  • step 2 After completing step 1, vortex the EP tube for 30 seconds, and briefly centrifuge the reaction solution to the bottom of the EP tube.
  • step 3 place the EP tube in a mixer (the mixer has been preheated to 41°C) and incubate with shaking at 41°C and 1400rpm for 30 minutes to obtain the hybridization product (i.e., the entire system in the EP tube). , containing magnetic beads).
  • Blocking buffer Add casein to PBS buffer to a concentration of 1 mg/ml.
  • step 5 Take the 96-well flat-bottomed plate that has completed step 4 and place it on a magnetic stand. Add 50 ⁇ L of the hybridization product obtained in step 3 to each reaction well, wash the magnetic beads three times with blocking buffer, and aspirate the waste liquid.
  • step 5 take the 96-well flat-bottom plate, add 50 ⁇ L SA-PE solution to each reaction well, then seal the 96-well plate with tin foil to avoid light, and incubate with shaking at 400 rpm at room temperature for 20 minutes.
  • SA-PE solution Dilute streptavidin phycoerythrin (SA-PE) (Abcam, product number: ab239759) with SA-PE diluent so that the concentration of SA-PE is 3 ⁇ g/mL.
  • SA-PE diluent contains 2.0M tetramethylammonium chloride (TMAC), 75mM trishydroxymethylaminomethane hydrochloride (Tris-HCl), 6mM ethylenediaminetetraacetic acid (EDTA), 1.5g/L Sarkosyl, 1.0g/L casein, and the balance is water.
  • step 6 wash the magnetic beads three times with blocking buffer and discard the supernatant.
  • step 7 use a degaussing instrument to degauss the 96-well flat-bottomed plate, then fill the reaction wells with blocking buffer and let stand for 10 minutes until the magnetic beads settle to the bottom.
  • step 8 use a fluorescence camera to collect the following information: identify the barcode on the digital magnetic beads under the white light channel, collect the fluorescence signal of the probe under the fluorescence channel, and perform data analysis.
  • a fragment of approximately 2000 bp in the L1 region of HPV16 virus in cervical exfoliated cell specimens was amplified by PCR (after sequencing, this fragment was the target sequence of the above primer pair in the HPV16 full genome; the HPV16 full genome sequence is recorded at https:// pave.niaid.nih.gov/database, whose ID is gi
  • HPV18F 5’-3’
  • ATGTGCCTGTATACA ATGTGCCTGTATACA
  • HPV18R 5’-3’
  • TTACTTCCTTGGCACGTACAC TTACTTCCTTGGCACGTACAC
  • a fragment of approximately 2000 bp in the L1 region of the HPV18 virus in cervical exfoliated cell specimens was amplified by PCR (after sequencing, this fragment was the target sequence of the above primer pair in the HPV18 full genome; the HPV18 full genome sequence is recorded at https:// pave.niaid.nih.gov/database, whose ID is gi
  • test standards are HPV16 standard and HPV18 standard.
  • the target concentrations are 20,000 copies/ ⁇ l, 2,000 copies/ ⁇ l, or 200 copies/ ⁇ l.
  • equal volumes of HPV16 standard diluent and HPV18 standard diluent of equal copy numbers were mixed to obtain a mixed standard diluent.
  • the HPV16 target concentration and HPV18 target concentration are both 10,000 copies/ ⁇ l.
  • the HPV16 target concentration and HPV18 target concentration are both 1000 copies/ ⁇ l.
  • the HPV16 target concentration and HPV18 target concentration are both 100 copies/ ⁇ l.
  • Example 2 Use the mixed standard dilution as a template and operate according to step 1 in Example 2 to obtain 50 ⁇ l of PCR product. Set up an equal volume of TE buffer instead of the mixed standard diluent as a blank control.
  • the fluorescence value captured by the HPV16 probe was used as the signal value, and the fluorescence value captured by the HPV18 probe was used as the background fluorescence (noise) value.
  • the fluorescence value captured by the HPV18 probe was used as the signal value, and the fluorescence value captured by the HPV16 probe was used as the background fluorescence (noise) value.
  • Signal-to-noise ratio signal value/noise value.
  • the invention discloses a universal joint detection method of different DNA targets using digital magnetic beads.
  • the core idea is to realize target detection through specific targeted amplification primers and corresponding capture probes.
  • this technology is only used for the detection of different subtypes of HPV.
  • any nucleic acid target can be jointly detected using this solution by designing specific primers and probes.
  • this technology can be used to screen multiple sources of respiratory tract infections, gastroenteritis infections, etc.

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Abstract

The present invention relates to a method for detecting multiple target nucleic acids, and in particular, to a method for achieving rapid detection and simultaneous typing of various pathogenic microorganisms by utilizing digital magnetic beads. Specifically, provided is a method for detecting n target nucleic acids by utilizing bar codes unique to digital magnetic beads to achieve simultaneous detection of multiple targets. By taking HPV as an example, the co-detection of HPV16 and HPV18 is achieved, which can be extended to the co-detection of multiple targets, for example, the co-detection of more than ten subtypes and even hundreds of subtypes of HPV.

Description

一种多目标核酸的检测方法A detection method for multi-target nucleic acids
本发明提供了一种多目标核酸的检测方法，具体涉及利用数码磁珠实现对多种病原微生物的快速检测以及同时分型的方法。The invention provides a multi-target nucleic acid detection method, specifically involving a method of using digital magnetic beads to achieve rapid detection and simultaneous typing of multiple pathogenic microorganisms.
背景技术Background technique
人乳头瘤病毒(HPV)属于乳多空病毒科的乳头瘤空泡病毒A属，为球形DNA病毒，其能引起人体皮肤黏膜的鳞状上皮增殖。现今已知的HPV亚型多达百种，而其中有几十种已经证明会引起人类不适症状，例如寻常疣、生殖器疣(尖锐湿疣)等症状。高危型HPV甚至能够引起宫颈癌、肛门癌、阴道癌、口鼻梁癌、外阴癌、阴茎癌等多种恶性肿瘤，其中又以宫颈癌为甚。Human papillomavirus (HPV) belongs to the genus Papillomavirus A of the family Papovaviridae. It is a spherical DNA virus that can cause the proliferation of squamous epithelium in human skin and mucous membranes. There are hundreds of HPV subtypes known today, and dozens of them have been proven to cause uncomfortable symptoms in humans, such as common warts and genital warts (genital warts). High-risk HPV can even cause a variety of malignant tumors such as cervical cancer, anal cancer, vaginal cancer, mouth and nose cancer, vulvar cancer, and penile cancer, among which cervical cancer is the most common.
开展HPV筛查可以有效降低宫颈癌的发病率和死亡率。北美、澳大利亚及欧洲等有完善筛查制度的国家或地区的经验证明，HPV筛查可以有效降低宫颈癌的发病率和死亡率，而开展大规模宫颈癌筛查对提高人类生存质量具有重要的推动作用。Carrying out HPV screening can effectively reduce the incidence and mortality of cervical cancer. The experience of countries or regions with complete screening systems such as North America, Australia and Europe has proven that HPV screening can effectively reduce the incidence and mortality of cervical cancer, and large-scale cervical cancer screening is important for improving the quality of human life. Promoting effect.
现有技术中HPV分型多采用实时荧光定量PCR(qPCR)来实现。该方案中，反应体系中加入与探针偶联的荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析。标记有荧光素的Taqman探针与模板DNA混合后，完成高温变性、低温复性、适温延伸的热循环，并遵守聚合酶链反应规律，与模板DNA互补配对的Taqman探针被切断，荧光素游离于反应体系中，在特定光激发下发出荧光；随着循环次数的增加，被扩增的目的基因片段呈指数规律增长，通过实时检测与之对应的随扩增而变化荧光信号强度，求得Ct值，同时利用数个已知模板浓度的标准品作对照，即可得出待测标本目的基因的拷贝数。然而利用此方案能同时区分的HPV亚型是有限的。In the existing technology, HPV typing is mostly achieved by real-time fluorescence quantitative PCR (qPCR). In this solution, a fluorescent group coupled to a probe is added to the reaction system, and the entire PCR process is monitored in real time using fluorescence signal accumulation. Finally, the unknown template is quantitatively analyzed through a standard curve. After the Taqman probe labeled with fluorescein is mixed with the template DNA, a thermal cycle of high-temperature denaturation, low-temperature renaturation, and appropriate temperature extension is completed, and the rules of the polymerase chain reaction are followed. The Taqman probe complementary to the template DNA is cut off, and the fluorescence The protein is free in the reaction system and emits fluorescence under specific light excitation; as the number of cycles increases, the amplified target gene fragment grows exponentially, and through real-time detection of the corresponding changes in fluorescence signal intensity with amplification, Calculate the Ct value and use several standards with known template concentrations as controls to determine the copy number of the target gene in the sample to be tested. However, the HPV subtypes that can be distinguished simultaneously using this scheme are limited.
由此开发一种能快速对HPV进行多亚型同时分型的方案，对于开展宫颈癌筛查以提高人类生存质量具有重要的推动作用。The development of a program that can quickly classify multiple HPV subtypes simultaneously will play an important role in promoting cervical cancer screening to improve the quality of human life.
发明公开invention disclosure
本发明提供了一种多目标核酸的检测方法，具体涉及利用数码磁珠实现对多种病原微生物的快速检测以及同时分型的方法。The invention provides a multi-target nucleic acid detection method, specifically involving a method of using digital magnetic beads to achieve rapid detection and simultaneous typing of multiple pathogenic microorganisms.
本发明提供了一种用于检测n种目标核酸的方法，包括如下步骤：The invention provides a method for detecting n target nucleic acids, which includes the following steps:
(1)将待测核酸进行扩增反应；所述扩增反应的目的为：当待测核酸中含有目标核酸时，通过扩增反应获得末端修饰有基团甲的扩增产物；(1) The nucleic acid to be tested is subjected to an amplification reaction; the purpose of the amplification reaction is: when the nucleic acid to be tested contains a target nucleic acid, an amplification product with a terminal modified with group A is obtained through the amplification reaction;
(2)将所述扩增反应的产物与n种偶联探针的数码磁珠进行杂交；每种偶联探针的数码磁珠可以结合一种目标核酸分子；偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将一种用于捕获目标核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；(2) Hybridize the product of the amplification reaction with n digital magnetic beads coupled to probes; each digital magnetic bead coupled to the probe can bind to one target nucleic acid molecule; the digital magnetic beads coupled to the probe Beads are obtained by coupling a probe used to capture target nucleic acids and digital magnetic beads; each digital magnetic bead coupled to a probe is obtained by coupling a probe used to capture target nucleic acids and a digital magnetic bead. Obtained; each type of digital magnetic beads has the same barcode, and different types of digital magnetic beads have different barcodes;
(3)将所述杂交的产物与检测元件进行反应；检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；(3) React the hybridized product with a detection element; the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
(4)将所述反应后的体系进行如下检测：检测磁珠的条形码以及光信号，根据条形码和光信号确定待测核酸中是否含有目标核酸和/或确定待测核酸中含有n种目标核酸中的哪一种或者哪几种；(4) The reaction system is tested as follows: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains the target nucleic acid and/or determine whether the nucleic acid to be tested contains n kinds of target nucleic acids based on the barcode and light signal. Which type or types;
n为2以上的自然数。n is a natural number above 2.
具体的，所述步骤(1)中，所述扩增反应为非对称PCR扩增反应。Specifically, in step (1), the amplification reaction is an asymmetric PCR amplification reaction.
非对称PCR扩增反应的目的为：当待测核酸中含有目标核酸时，通过扩增反应获得末端修饰有基团甲的单链扩增产物。The purpose of the asymmetric PCR amplification reaction is: when the nucleic acid to be tested contains the target nucleic acid, a single-stranded amplification product with a group A modified at the end is obtained through the amplification reaction.
具体的，所述非对称PCR扩增反应，扩增体系中上游引物和下游引物的浓度不同。Specifically, in the asymmetric PCR amplification reaction, the concentrations of the upstream primer and the downstream primer in the amplification system are different.
具体的，所述非对称PCR扩增反应，扩增体系中上游引物和下游引物的摩尔配比可为0.1:10。Specifically, in the asymmetric PCR amplification reaction, the molar ratio of the upstream primer and the downstream primer in the amplification system can be 0.1:10.
具体的，所述非对称PCR扩增反应的反应体系如表1所示。Specifically, the reaction system of the asymmetric PCR amplification reaction is shown in Table 1.
具体的，所述非对称PCR扩增反应的反应程序如表2所示。Specifically, the reaction procedure of the asymmetric PCR amplification reaction is shown in Table 2.
所述步骤(1)中，所述扩增反应采用的引物对由上游引物和下游引物组成；下游引物的5'末端修饰有所述基团甲。In the step (1), the primer pair used in the amplification reaction consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with the group A.
示例性的，所述基团甲和基团乙互为亲和基团。For example, the group A and the group B are affinity groups for each other.
示例性的，所述基团甲为生物素，优选TripleBiotin(又称为Triple-Biotin)。Illustratively, the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
示例性的，所述基团乙为链霉亲和素。Illustratively, the group B is streptavidin.
示例性的，所述光信号为荧光信号。Exemplarily, the light signal is a fluorescence signal.
示例性的，所述基团丙为藻红蛋白。Exemplarily, the group C is phycoerythrin.
示例性的，所述检测元件为链霉亲和素藻红蛋白(SA-PE)。Exemplarily, the detection element is streptavidin phycoerythrin (SA-PE).
示例性的，所述探针的5'末端具NH 2C 12修饰。 Exemplarily, the 5' end of the probe is modified with NH 2 C 12 .
对于本发明的具体实施例来说，所述n为2。For specific embodiments of the present invention, n is 2.
本发明提供的方法可用于多种病原微生物的联检。所述多种病原微生物包括但不限于如下：流感病毒、新冠病毒、痘病毒、腺病毒等，或者多种致病菌，例如，大肠杆菌、金黄葡萄球菌、结核杆菌、沙门氏菌以及链球菌等。The method provided by the invention can be used for joint detection of multiple pathogenic microorganisms. The various pathogenic microorganisms include but are not limited to the following: influenza virus, new coronavirus, poxvirus, adenovirus, etc., or a variety of pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Streptococcus, etc.
本发明提供的方法可用于多种生物标志物的联检。所述多种生物标志物包括但不限于如下：外周血中的肝癌标志物(例如甲胎蛋白)的特征性核酸、外周血中的结直肠癌标志物的特征性核酸等。The method provided by the invention can be used for joint detection of multiple biomarkers. The various biomarkers include, but are not limited to, the following: nucleic acids characteristic of liver cancer markers (such as alpha-fetoprotein) in peripheral blood, nucleic acids characteristic of colorectal cancer markers in peripheral blood, etc.
本发明还提供了一种用于检测n种目标核酸的试剂盒，包括由n个引物对组成的扩增引物组、由n种偶联探针的数码磁珠组成的数码磁珠组和检测元件；The invention also provides a kit for detecting n kinds of target nucleic acids, including an amplification primer set composed of n primer pairs, a digital magnetic bead set composed of n kinds of digital magnetic beads coupled to probes, and a detection kit. element;
扩增引物组中：不同引物对用于扩增不同的目标核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；In the amplification primer set: different primer pairs are used to amplify different target nucleic acids; each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
数码磁珠组中：偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将一种用于捕获目标核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；In the digital magnetic bead set: the digital magnetic beads for coupling probes are obtained by coupling the probe used to capture the target nucleic acid with the digital magnetic beads; each type of digital magnetic beads for coupling probes is obtained by coupling one type of digital magnetic beads for capturing target nucleic acids. The target nucleic acid probe is coupled to a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcodes;
检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；The detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
n为2以上的自然数。n is a natural number above 2.
具体的，每个引物对中，上游引物和下游引物的摩尔配比可为0.1:10。Specifically, in each primer pair, the molar ratio of the upstream primer and the downstream primer can be 0.1:10.
示例性的，所述基团甲和基团乙互为亲和基团。For example, the group A and the group B are affinity groups for each other.
示例性的，所述基团甲为生物素，优选为TripleBiotin(又称为Triple-Biotin)。Illustratively, the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
示例性的，所述基团乙为链霉亲和素。Illustratively, the group B is streptavidin.
示例性的，所述光信号为荧光信号。Exemplarily, the light signal is a fluorescence signal.
示例性的，所述基团丙为藻红蛋白。Exemplarily, the group C is phycoerythrin.
示例性的，所述检测元件为链霉亲和素藻红蛋白(SA-PE)。Exemplarily, the detection element is streptavidin phycoerythrin (SA-PE).
示例性的，所述探针的5'末端具NH 2C 12修饰。 Exemplarily, the 5' end of the probe is modified with NH 2 C 12 .
对于本发明的具体实施例来说，所述n为2。For specific embodiments of the present invention, n is 2.
本发明提供的试剂盒可用于多种病原微生物的联检。所述多种病原微生物包括但不限于如下：流感病毒、新冠病毒、痘病毒、腺病毒等，或者多种致病菌，例如，大肠杆菌、金黄葡萄球菌、结核杆菌、沙门氏菌以及链球菌等。The kit provided by the invention can be used for joint detection of multiple pathogenic microorganisms. The various pathogenic microorganisms include but are not limited to the following: influenza virus, new coronavirus, poxvirus, adenovirus, etc., or a variety of pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Streptococcus, etc.
本发明提供的试剂盒可用于多种生物标志物的联检。所述多种生物标志物包括但不限于如下：外周血中的肝癌标志物(例如甲胎蛋白)的特征性核酸、外周血中的结直肠癌标志物的特征性核酸等。The kit provided by the invention can be used for joint detection of multiple biomarkers. The various biomarkers include, but are not limited to, the following: nucleic acids characteristic of liver cancer markers (such as alpha-fetoprotein) in peripheral blood, nucleic acids characteristic of colorectal cancer markers in peripheral blood, etc.
本发明还提供了一种用于检测HPV的方法，包括如下步骤：The present invention also provides a method for detecting HPV, which includes the following steps:
(1)以待测核酸样本为模板，采用扩增引物组进行PCR扩增；扩增引物组由n个引物对组成；每个引物对用于扩增一种型别的HPV的特征性核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；(1) Use the nucleic acid sample to be tested as a template and use an amplification primer set to perform PCR amplification; the amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV ;Each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
(2)将所述PCR扩增的产物与数码磁珠组进行杂交；数码磁珠组由n种偶联探针的数码磁珠组成；偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将用于捕获一种型别的HPV的特征性核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；(2) Hybridize the PCR amplified product with a digital magnetic bead set; the digital magnetic bead set consists of n digital magnetic beads coupled to probes; the digital magnetic beads coupled to probes will be used to capture the target Nucleic acid probes are obtained by coupling to digital magnetic beads; each digital magnetic bead coupling probe is obtained by coupling a probe for capturing the characteristic nucleic acid of a type of HPV to a digital magnetic bead. ; Each type of digital magnetic beads has the same barcode, and different types of digital magnetic beads have different barcodes;
(3)将所述杂交的产物与检测元件进行反应；检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；(3) React the hybridized product with a detection element; the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
(4)将所述反应后的体系进行如下检测：检测磁珠的条形码以及光信号，根据条形码和光信号确定待测核酸中是否含有HPV和/或确定待测核酸中含有n种HPV中的哪一种或者哪几种；(4) The reaction system is subjected to the following detection: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains HPV and/or determine which of the n types of HPV is contained in the nucleic acid to be tested based on the barcode and light signal. One or several types;
n为2以上的自然数。n is a natural number above 2.
具体的，步骤(1)中，所述PCR扩增为非对称PCR扩增。Specifically, in step (1), the PCR amplification is asymmetric PCR amplification.
具体的，所述非对称PCR扩增，扩增体系中上游引物和下游引物的浓度不同。Specifically, in the asymmetric PCR amplification, the concentrations of the upstream primer and the downstream primer in the amplification system are different.
具体的，所述非对称PCR扩增，扩增体系中上游引物和下游引物的摩尔配比可为0.1:10。Specifically, in the asymmetric PCR amplification, the molar ratio of the upstream primer and the downstream primer in the amplification system can be 0.1:10.
具体的，所述非对称PCR扩增的反应体系如表1所示。Specifically, the reaction system of the asymmetric PCR amplification is shown in Table 1.
具体的，所述非对称PCR扩增的反应程序如表2所示。Specifically, the reaction procedure of the asymmetric PCR amplification is shown in Table 2.
示例性的，所述基团甲和基团乙互为亲和基团。For example, the group A and the group B are affinity groups for each other.
示例性的，所述基团甲为生物素，优选为TripleBiotin(又称为Triple-Biotin)。Illustratively, the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
示例性的，所述基团乙为链霉亲和素。Illustratively, the group B is streptavidin.
示例性的，所述光信号为荧光信号。Exemplarily, the light signal is a fluorescence signal.
示例性的，所述基团丙为藻红蛋白。Exemplarily, the group C is phycoerythrin.
示例性的，所述检测元件为链霉亲和素藻红蛋白(SA-PE)。Exemplarily, the detection element is streptavidin phycoerythrin (SA-PE).
示例性的，所述探针的5'末端具NH 2C 12修饰。 Exemplarily, the 5' end of the probe is modified with NH 2 C 12 .
对于本发明的具体实施例来说，所述n为2。For specific embodiments of the present invention, n is 2.
对于本发明的具体实施例来说，扩增引物组由2个引物对组成；一个引物对用于扩增HPV16的特征性核酸，由HPV16-F和HPV16-R组成；另一个引物对用于扩增HPV18的特征性核酸，由HPV18-F和HPV18-R组成。For specific embodiments of the present invention, the amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV16 Amplify the characteristic nucleic acid of HPV18, consisting of HPV18-F and HPV18-R.
对于本发明的具体实施例来说，数码磁珠组由2种偶联探针的数码磁珠组成；一种偶联探针的数码磁珠是将用于捕获HPV16的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV16的特征性核酸的探针命名为HPV16-P；另一种偶联探针的数码磁珠是将用于捕获HPV18的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV18的特征性核酸的探针命名为HPV18-P。For specific embodiments of the present invention, the digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is a probe that will be used to capture the characteristic nucleic acid of HPV16. The probe coupled to a digital magnetic bead for capturing the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupled to the probe is used to capture the characteristic nucleic acid of HPV18. The probe is coupled to a digital magnetic bead and is used to capture the characteristic nucleic acid of HPV18, named HPV18-P.
对于本发明的具体实施例来说：HPV16-F的核苷酸序列如SEQ ID NO：1所示；HPV16-R的核苷酸序列如SEQ ID NO：2所示；HPV16-P的核苷酸序列如SEQ ID NO：3所示；HPV18-F的核苷酸序列如SEQ ID NO：4所示；HPV18-R的核苷酸序列如SEQ ID NO：5所示；HPV18-P的核苷酸序列如SEQ ID NO：6所示。For specific embodiments of the present invention: the nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleoside of HPV16-P The acid sequence is shown in SEQ ID NO: 3; the nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleic acid sequence of HPV18-P is shown in SEQ ID NO: 4. The nucleotide sequence is shown in SEQ ID NO: 6.
本发明还提供了一种用于检测HPV的试剂盒，包括扩增引物组、数码磁珠组和检测元件；The invention also provides a kit for detecting HPV, including an amplification primer set, a digital magnetic bead set and a detection element;
扩增引物组由n个引物对组成；每个引物对用于扩增一种型别的HPV的特征性核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；The amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV; each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with Group A;
数码磁珠组由n种偶联探针的数码磁珠组成；偶联探针的数码磁珠是将 用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将用于捕获一种型别的HPV的特征性核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；The digital magnetic bead set consists of n types of digital magnetic beads coupled to probes; the digital magnetic beads coupled to probes are obtained by coupling the probe used to capture the target nucleic acid and the digital magnetic beads; each coupled probe The digital magnetic beads are obtained by coupling a probe used to capture the characteristic nucleic acid of a type of HPV with a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcode;
检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；The detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
n为2以上的自然数。n is a natural number above 2.
具体的，每个引物对中，上游引物和下游引物的摩尔配比可为0.1:10。Specifically, in each primer pair, the molar ratio of the upstream primer and the downstream primer can be 0.1:10.
示例性的，所述基团甲和基团乙互为亲和基团。For example, the group A and the group B are affinity groups for each other.
示例性的，所述基团甲为生物素，优选为TripleBiotin(又称为Triple-Biotin)。Illustratively, the group A is biotin, preferably TripleBiotin (also known as Triple-Biotin).
示例性的，所述基团乙为链霉亲和素。Illustratively, the group B is streptavidin.
示例性的，所述光信号为荧光信号。Exemplarily, the light signal is a fluorescence signal.
示例性的，所述基团丙为藻红蛋白。Exemplarily, the group C is phycoerythrin.
示例性的，所述检测元件为链霉亲和素藻红蛋白(SA-PE)。Exemplarily, the detection element is streptavidin phycoerythrin (SA-PE).
示例性的，所述探针的5'末端具NH 2C 12修饰。 Exemplarily, the 5' end of the probe is modified with NH 2 C 12 .
对于本发明的具体实施例来说，所述n为2。For specific embodiments of the present invention, n is 2.
对于本发明的具体实施例来说，扩增引物组由2个引物对组成；一个引物对用于扩增HPV16的特征性核酸，由HPV16-F和HPV16-R组成；另一个引物对用于扩增HPV18的特征性核酸，由HPV18-F和HPV18-R组成。For specific embodiments of the present invention, the amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV16 Amplify the characteristic nucleic acid of HPV18, consisting of HPV18-F and HPV18-R.
对于本发明的具体实施例来说，数码磁珠组由2种偶联探针的数码磁珠组成；一种偶联探针的数码磁珠是将用于捕获HPV16的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV16的特征性核酸的探针命名为HPV16-P；另一种偶联探针的数码磁珠是将用于捕获HPV18的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV18的特征性核酸的探针命名为HPV18-P。For specific embodiments of the present invention, the digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is a probe that will be used to capture the characteristic nucleic acid of HPV16. The probe coupled to a digital magnetic bead for capturing the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupled to the probe is used to capture the characteristic nucleic acid of HPV18. The probe is coupled to a digital magnetic bead and is used to capture the characteristic nucleic acid of HPV18, named HPV18-P.
对于本发明的具体实施例来说：HPV16-F的核苷酸序列如SEQ ID NO：1所示；HPV16-R的核苷酸序列如SEQ ID NO：2所示；HPV16-P的核苷酸序列如SEQ ID NO：3所示；HPV18-F的核苷酸序列如SEQ ID NO：4所示；HPV18-R的核苷酸序列如SEQ ID NO：5所示；HPV18-P的核苷酸序列如SEQ ID NO：6所示。For specific embodiments of the present invention: the nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleoside of HPV16-P The acid sequence is shown in SEQ ID NO: 3; the nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleic acid sequence of HPV18-P is shown in SEQ ID NO: 4. The nucleotide sequence is shown in SEQ ID NO: 6.
对于本发明的具体实施例来说，偶联探针的数码磁珠的制备方法具体如 实施例2的步骤二所述。For specific embodiments of the present invention, the preparation method of digital magnetic beads coupled to probes is specifically as described in step 2 of Example 2.
对于本发明的具体实施例来说，将所述杂交的产物与检测元件进行反应的方法具体如实施例2的步骤三的步骤1至6所述。For specific embodiments of the present invention, the method for reacting the hybridization product with the detection element is specifically as described in steps 1 to 6 of step three of embodiment 2.
对于本发明的具体实施例来说，将所述杂交的产物与检测元件进行反应的方法具体如实施例2的步骤三的步骤1至8所述。For specific embodiments of the present invention, the method for reacting the hybridization product with the detection element is specifically as described in Steps 1 to 8 of Step 3 of Embodiment 2.
对于本发明的具体实施例，所示链霉亲和素藻红蛋白具体可为：Abcam,货号ab239759。For specific embodiments of the present invention, the streptavidin phycoerythrin shown can specifically be: Abcam, product number ab239759.
对于本发明的具体实施例，所述数码磁珠具体可为台湾稳银生产的数码磁珠。For specific embodiments of the present invention, the digital magnetic beads may be digital magnetic beads produced by Taiwan Wen Yin.
现有技术的缺点：现有技术方案无法实现靶标的联检，导致成本高，操作总时间长。本发明以HPV为示例，实现了HPV16与HPV18的联检，事实上，本发明的方案可以推及至多靶标联检。例如，十几种亚型甚至是上百种亚型的HPV联检。Disadvantages of the existing technology: The existing technical solution cannot achieve joint detection of targets, resulting in high costs and long total operation time. Taking HPV as an example, the present invention realizes the joint detection of HPV16 and HPV18. In fact, the solution of the present invention can be extended to joint detection of multiple targets. For example, joint testing of more than a dozen or even hundreds of subtypes of HPV.
本发明技术方案带来的有益效果：①本发明中数码磁珠的多种编码性能实现多靶标的联检，这一特性可实现单一样品多核酸靶标的单孔检测(也就是HPV多亚型的单孔同时检测)，可提高检测效率，极大的降低的工作量并降低了试剂成本；②在DNA靶标扩增过程中采用了非对称PCR方案使得待测DNA直接是单链DNA，从而略去了高温变性以及低温孵育过程，简化了实验步骤的同时，避免了高温可能产生气溶胶使样本交叉污染的问题。Beneficial effects brought by the technical solution of the present invention: ① The multiple encoding properties of the digital magnetic beads in the present invention enable joint detection of multiple targets. This feature enables single-hole detection of multiple nucleic acid targets in a single sample (that is, detection of multiple HPV subtypes). Single-well simultaneous detection) can improve detection efficiency, greatly reduce workload and reduce reagent costs; ② In the DNA target amplification process, an asymmetric PCR scheme is used to make the DNA to be tested directly single-stranded DNA, thereby omitting The high-temperature denaturation and low-temperature incubation processes are eliminated, which simplifies the experimental steps and avoids the problem of cross-contamination of samples due to aerosols generated by high temperatures.
附图说明Description of drawings
图1为利用数码磁珠完成核酸检测的通用流程。Figure 1 shows the general process for nucleic acid detection using digital magnetic beads.
图2为联检流程图。Figure 2 is a joint inspection flow chart.
图3为采用本联检技术实现HPV16、HPV18的联合检测所得HPV16、HPV18的信号噪音比。Figure 3 shows the signal-to-noise ratio of HPV16 and HPV18 obtained by joint detection of HPV16 and HPV18 using this joint detection technology.
实施发明的最佳方式Best way to implement your invention
以下的实施例便于更好地理解本发明，但并不限定本发明。The following examples facilitate a better understanding of the present invention, but do not limit the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。实施例中的定量试验，如无特殊说明，均设置至少三次重复，结果取平均值。The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified. Unless otherwise specified, the quantitative tests in the examples were repeated at least three times, and the results were averaged.
如无特殊说明，实施例中的DNA分子方向均为5’→3'方向。Unless otherwise specified, the direction of DNA molecules in the examples is 5'→3' direction.
实施例1、引物探针的设计Example 1. Design of primers and probes
通过大量预实验和效果验证，筛选到效果最佳的引物探针。Through extensive pre-experiments and effect verification, the primers and probes with the best results were selected.
用于检测HPV16的引物和探针如下：The primers and probes used to detect HPV16 are as follows:
HPV16-F(上游引物)：GCACAGGGCCACAATAATGG；HPV16-F (upstream primer): GCACAGGGCCACAATAATGG;
HPV16-R(下游引物)：TripleBiotin-CATAACGTCTGCAGTTAAGGTTATT；HPV16-R (downstream primer): TripleBiotin-CATAACGTCTGCAGTTAAGGTTATT;
HPV16-P(探针)：NH 2C 12-TGTGCTGCCATATCTACTTCAGA。 HPV16-P (probe): NH 2 C 12 -TGTGCTGCCATATCTACTTCAGA.
用于检测HPV18的引物和探针如下：The primers and probes used to detect HPV18 are as follows:
HPV18-F(上游引物)：GCACAGGGTCATAACAATGG；HPV18-F (upstream primer): GCACAGGGTCATAACAATGG;
HPV18-R(下游引物)：TripleBiotin-CATGTCTGCTATACTGCTTAAAT；HPV18-R (downstream primer): TripleBiotin-CATGTTCTGCTATACTGCTTAAAT;
HPV18-P(探针)：NH 2C 12-AGTCTCCTGTACCTGGGCAA。 HPV18-P (probe): NH 2 C 12 -AGTCTCCTGTACCTGGGCAA.
以上引物探针均为单链DNA分子，且方向均为5'→3'。The above primer probes are all single-stranded DNA molecules, and the direction is 5'→3'.
下游引物中，5’末端修饰有TripleBiotin(又称为Triple-Biotin)。In the downstream primer, the 5' end is modified with TripleBiotin (also known as Triple-Biotin).
探针中，5’末端修饰有NH 2C 12。 In the probe, the 5' end is modified with NH 2 C 12 .
HPV16-F的核苷酸序列如SEQ ID NO：1所示。HPV16-R的核苷酸序列如SEQ ID NO：2所示。HPV16-P的核苷酸序列如SEQ ID NO：3所示。The nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1. The nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2. The nucleotide sequence of HPV16-P is shown in SEQ ID NO: 3.
HPV18-F的核苷酸序列如SEQ ID NO：4所示。HPV18-R的核苷酸序列如SEQ ID NO：5所示。HPV18-P的核苷酸序列如SEQ ID NO：6所示。The nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4. The nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5. The nucleotide sequence of HPV18-P is shown in SEQ ID NO: 6.
实施例2、方法的建立Example 2. Establishment of method
一、目标产物的扩增1. Amplification of target product
1、制备表1所示的PCR反应体系。1. Prepare the PCR reaction system shown in Table 1.
表1中，下游引物为HPV16-R和HPV18-R。In Table 1, the downstream primers are HPV16-R and HPV18-R.
表1中，上游引物为HPV16-F和HPV18-F。In Table 1, the upstream primers are HPV16-F and HPV18-F.
表1Table 1
组分Components 体积(μL)Volume(μL)
KAPA2G Fast Multiplex MixKAPA2G Fast Multiplex Mix 2525
下游引物(每种下游引物的浓度均为10μM)Downstream primers (the concentration of each downstream primer is 10 μM) 55
上游引物(每种上游引物的浓度均为0.1μM)Upstream primers (the concentration of each upstream primer is 0.1 μM) 1010
H 2O H 2 O 99
模板 template 11
总体积total capacity 5050
KAPA2G Fast Multiplex Mix：美国KAPA Biosystems公司，产品目录号为KK5802。KAPA2G Fast Multiplex Mix: American KAPA Biosystems company, product catalog number is KK5802.
2、完成步骤1后，瞬时离心，将液体收集于PCR管底部。2. After completing step 1, centrifuge briefly and collect the liquid at the bottom of the PCR tube.
3、按照表2进行PCR反应，完成反应的体系即为PCR产物。3. Carry out PCR reaction according to Table 2. The system that completes the reaction is the PCR product.
表2Table 2
Figure PCTCN2022111673-appb-000001
Figure PCTCN2022111673-appb-000001
二、将数码磁珠与探针偶联，得到偶联探针的数码磁珠。2. Couple the digital magnetic beads with the probe to obtain the digital magnetic beads coupled to the probe.
数码磁珠：数码磁珠采购自台湾稳银(winmems，网址：Digital magnetic beads: Digital magnetic beads are purchased from Winmems, Taiwan, website:
https://www.winmemstech.com/cn/page/custom28/)。数码磁珠上蚀刻有不同的能被白光识别的条形码；不同编号的磁珠混在一起时，能通过条形码进行识别分类。数码磁珠4℃保存。数码磁珠由基质和磁性物质组成(基质为聚苯乙烯，磁性物质为四氧化三铁)，外表面修饰有羧基并且外表面采用光刻法蚀刻有不同的能被白光识别的条形码；磁珠外形为长方形，其规格为25*35mm。https://www.winmemstech.com/cn/page/custom28/). Digital magnetic beads are etched with different barcodes that can be identified by white light; when magnetic beads with different numbers are mixed together, they can be identified and classified through the barcodes. Digital magnetic beads are stored at 4°C. Digital magnetic beads are composed of a matrix and magnetic material (the matrix is polystyrene and the magnetic material is ferroferric oxide). The outer surface is modified with carboxyl groups and the outer surface is etched with different barcodes that can be recognized by white light using photolithography. Magnetic beads The shape is rectangular and its specification is 25*35mm.
本步骤中，每种探针分别与具有特定条形码的数码磁珠进行偶联反应，从而使每种探针具有特定的条形码。探针为实施例1中的HPV16-P或HPV18-P。HPV16-P特异性识别HPV16。HPV18-P特异性识别HPV18-P。In this step, each probe is coupled to digital magnetic beads with a specific barcode, so that each probe has a specific barcode. The probe is HPV16-P or HPV18-P in Example 1. HPV16-P specifically recognizes HPV16. HPV18-P specifically recognizes HPV18-P.
每种探针分别和一种数码磁珠(一种数码磁珠指的是数码磁珠具有相同的条形码)偶联，分别进行如下步骤：Each probe is coupled to a digital magnetic bead (a digital magnetic bead refers to a digital magnetic bead with the same barcode), and the following steps are performed:
1、取50μL数码磁珠，加入至1.5mL EP管中。1. Take 50μL of digital magnetic beads and add it to a 1.5mL EP tube.
2、完成步骤1后，在所述EP管中加入500μL异丙醇，涡旋振荡30s以充分混合。2. After completing step 1, add 500 μL isopropyl alcohol to the EP tube and vortex for 30 seconds to mix thoroughly.
3、完成步骤2后，所述EP管离心30s，使磁珠尽量沉降以利于磁力架吸附。3. After completing step 2, centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
4、完成步骤3后，所述EP管置于磁力架上静止2min以使数码磁珠悉数沉淀。4. After completing step 3, place the EP tube on a magnetic stand for 2 minutes to allow all digital magnetic beads to precipitate.
5、完成步骤4后，保持所述EP管在磁力架上，尽量吸弃上清溶液。5. After completing step 4, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
6、完成步骤5后，在所述EP管中加入500μL清洗缓冲液1，涡旋振荡30s以充分混合。6. After completing step 5, add 500 μL of washing buffer 1 to the EP tube and vortex for 30 seconds to mix thoroughly.
清洗缓冲液1(pH5.0)：含0.1M 2-(N-吗啉)乙磺酸(MES)和0.05％吐温20(Tween 20)，余量为水。Washing buffer 1 (pH5.0): contains 0.1M 2-(N-morpholine)ethanesulfonic acid (MES) and 0.05% Tween 20 (Tween 20), and the balance is water.
7、完成步骤6后，所述EP管离心30s，使磁珠尽量沉降以利于磁力架吸附。7. After completing step 6, centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
8、完成步骤7后，保持所述EP管置于磁力架上静止2min以使数码磁珠悉数沉淀。8. After completing step 7, keep the EP tube on the magnetic stand for 2 minutes to allow all digital magnetic beads to settle.
9、完成步骤8后，保持所述EP管在磁力架上，尽量吸弃上清溶液。9. After completing step 8, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
10、将步骤6-9重复一次。10. Repeat steps 6-9 once.
11、完成步骤10后，在所述EP管中加入500μL清洗缓冲液2，涡旋振荡30s以充分混合。11. After completing step 10, add 500 μL of washing buffer 2 to the EP tube and vortex for 30 seconds to mix thoroughly.
清洗缓冲液2(pH5.0)：含0.1M 2-(N-吗啉)乙磺酸(MES)，余量为水。Washing buffer 2 (pH 5.0): contains 0.1M 2-(N-morpholine)ethanesulfonic acid (MES), and the balance is water.
12、完成步骤11后，所述EP管离心30s，使磁珠尽量沉降以利于磁力架吸附。12. After completing step 11, centrifuge the EP tube for 30 seconds to allow the magnetic beads to settle as much as possible to facilitate adsorption by the magnetic stand.
13、完成步骤12后，所述EP管置于磁力架上静止2min以使数码磁珠悉数沉淀。13. After completing step 12, place the EP tube on a magnetic stand for 2 minutes to allow all digital magnetic beads to precipitate.
14、完成步骤13后，保持EP管在磁力架上，尽量吸弃上清溶液。14. After completing step 13, keep the EP tube on the magnetic stand and try to absorb and discard the supernatant solution.
15、完成步骤14后，在所述EP管中加入28μL清洗缓冲液2、15μL EDC溶液、3μL NHS溶液和4μL 100mM探针溶液，充分混合。15. After completing step 14, add 28μL cleaning buffer 2, 15μL EDC solution, 3μL NHS solution and 4μL 100mM probe solution to the EP tube, and mix thoroughly.
EDC溶液的制备方法：将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶于清洗缓冲液2，使EDC的浓度为20mg/mL。现用现配。Preparation method of EDC solution: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in washing buffer 2 to make the concentration of EDC 20 mg/mL. Ready for use.
NHS溶液的制备方法：将N-羟基琥珀酰亚胺(NHS)溶于清洗缓冲液2，使NHS的浓度为20mg/mL。现用现配。Preparation method of NHS solution: Dissolve N-hydroxysuccinimide (NHS) in washing buffer 2 to make the concentration of NHS 20 mg/mL. Ready for use.
16、完成步骤15后，将所述EP管置于混匀仪中，1400rpm室温避光反应30min。16. After completing step 15, place the EP tube in a mixer and react at 1400 rpm at room temperature for 30 minutes in the dark.
17、完成步骤16后，用0.2g/L吐温-20(Tween-20)水溶液清洗磁珠一次，吸弃上清。17. After completing step 16, wash the magnetic beads once with 0.2g/L Tween-20 aqueous solution, and discard the supernatant.
18、完成步骤17后，用1g/L十二烷基硫酸钠(SDS)水溶液清洗磁珠一次，吸弃上清。18. After completing step 17, wash the magnetic beads once with 1g/L sodium dodecyl sulfate (SDS) aqueous solution, and discard the supernatant.
19、完成步骤18后，在所述EP管中加入100μL TE buffer，即为偶联探 针的数码磁珠溶液，4℃存储。19. After completing step 18, add 100 μL TE buffer, which is the digital magnetic bead solution of the coupled probe, into the EP tube and store at 4°C.
通过上述步骤，分别得到偶联HPV16-P的数码磁珠溶液和偶联HPV18-P的数码磁珠溶液。Through the above steps, the digital magnetic bead solution coupled to HPV16-P and the digital magnetic bead solution coupled to HPV18-P were obtained respectively.
三、目标产物与探针杂交检测荧光信号3. Hybridization of target product and probe to detect fluorescence signal
1、取EP管，加入33μL杂交缓冲液、10μL步骤一得到的PCR产物、1μL步骤二制备的偶联HPV16-P的数码磁珠溶液、1μL步骤二制备的偶联HPV18-P的数码磁珠溶液、5μL TE buffer。1. Take an EP tube and add 33 μL of hybridization buffer, 10 μL of the PCR product obtained in step one, 1 μL of the HPV16-P coupled digital magnetic bead solution prepared in step two, and 1 μL of the HPV18-P coupled digital magnetic beads prepared in step two. Solution, 5μL TE buffer.
杂交缓冲液(pH8.0)：含0.15M四甲基氯化铵(TMAC)、75mM三羟甲基氨基甲烷盐酸盐(Tris-HCl)、6mM乙二胺四乙酸(EDTA)、1.5g/L十二烷基肌氨酸钠(Sarkosyl)，余量为水。Hybridization buffer (pH8.0): Contains 0.15M tetramethylammonium chloride (TMAC), 75mM tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 6mM ethylenediaminetetraacetic acid (EDTA), 1.5g /L sodium lauryl sarcosinate (Sarkosyl), the balance is water.
2、完成步骤1后，将所述EP管涡旋震荡30s，瞬时离心将反应液离心至EP管底部。2. After completing step 1, vortex the EP tube for 30 seconds, and briefly centrifuge the reaction solution to the bottom of the EP tube.
3、完成步骤2后，将所述EP管置于混匀仪(混匀仪已预先预热至41℃)中，41℃、1400rpm震荡孵育30min，得到杂交产物(即EP管里的整个体系，含磁珠)。3. After completing step 2, place the EP tube in a mixer (the mixer has been preheated to 41°C) and incubate with shaking at 41°C and 1400rpm for 30 minutes to obtain the hybridization product (i.e., the entire system in the EP tube). , containing magnetic beads).
4、取96孔平底板，用封闭缓冲液润洗平衡，然后吸弃废液。4. Take a 96-well flat-bottom plate, rinse and balance it with blocking buffer, and then aspirate the waste liquid.
封闭缓冲液：将酪蛋白(casein)加入至PBS缓冲液中，使其浓度为1mg/ml。Blocking buffer: Add casein to PBS buffer to a concentration of 1 mg/ml.
5、取完成步骤4的96孔平底板，置于磁力架上，每个反应孔中加入50μL步骤3得到的杂交产物，用封闭缓冲液清洗磁珠三次，吸弃废液。5. Take the 96-well flat-bottomed plate that has completed step 4 and place it on a magnetic stand. Add 50 μL of the hybridization product obtained in step 3 to each reaction well, wash the magnetic beads three times with blocking buffer, and aspirate the waste liquid.
6、完成步骤5后，取所述96孔平底板，每个反应孔中加入50μL SA-PE溶液，然后用锡箔纸将96孔板密封避光，400rpm室温震荡孵育20min。6. After completing step 5, take the 96-well flat-bottom plate, add 50 μL SA-PE solution to each reaction well, then seal the 96-well plate with tin foil to avoid light, and incubate with shaking at 400 rpm at room temperature for 20 minutes.
SA-PE溶液的制备方法：将链霉亲和素藻红蛋白(SA-PE)(Abcam,货号：ab239759)利用SA-PE稀释液稀释，使SA-PE的浓度为3μg/mL。Preparation method of SA-PE solution: Dilute streptavidin phycoerythrin (SA-PE) (Abcam, product number: ab239759) with SA-PE diluent so that the concentration of SA-PE is 3 μg/mL.
SA-PE稀释液(pH8.0)：含2.0M四甲基氯化铵(TMAC)、75mM三羟甲基氨基甲烷盐酸盐(Tris-HCl)、6mM乙二胺四乙酸(EDTA)、1.5g/L十二烷基肌氨酸钠(Sarkosyl)、1.0g/L酪蛋白(casein)，余量为水。SA-PE diluent (pH8.0): contains 2.0M tetramethylammonium chloride (TMAC), 75mM trishydroxymethylaminomethane hydrochloride (Tris-HCl), 6mM ethylenediaminetetraacetic acid (EDTA), 1.5g/L Sarkosyl, 1.0g/L casein, and the balance is water.
7、完成步骤6后，用封闭缓冲液清洗磁珠三次，吸弃上清。7. After completing step 6, wash the magnetic beads three times with blocking buffer and discard the supernatant.
8、完成步骤7后，利用消磁仪对所述96孔平底板进行消磁，然后在反应孔中加满封闭缓冲液，静止10min以待磁珠沉降至底部。8. After completing step 7, use a degaussing instrument to degauss the 96-well flat-bottomed plate, then fill the reaction wells with blocking buffer and let stand for 10 minutes until the magnetic beads settle to the bottom.
9、完成步骤8后，利用荧光拍照仪采集如下信息：白光通道下识别数码磁珠上的条形码，荧光通道下采集探针的荧光信号并进行数据分析。9. After completing step 8, use a fluorescence camera to collect the following information: identify the barcode on the digital magnetic beads under the white light channel, collect the fluorescence signal of the probe under the fluorescence channel, and perform data analysis.
实施例3、方法的应用Example 3. Application of method
HPV16标准品的详细制备过程可参考文献“人乳头瘤病毒基因分型质控品的建立”。大致过程如下：设计HPV16L1目标区域扩增的上下游引物，引物序列分别为：HPV16F(5’-3’)，ATGCAGGTGACTTTTATTTACATC；HPV16R(5’-3’)，TTACAGCTTACGTTTTTTGCGTTT。通过PCR扩增宫颈脱落细胞标本中HPV16病毒L1区域中的一个长度约为2000bp的片段(经测序，该片段为HPV16全基因组中上述引物对的靶序列；HPV16全基因组序列记载于https://pave.niaid.nih.gov/数据库，其ID为gi|333031|lcl|HPV16REF)，然后将其插入pMD18-T质粒构建重组质粒。For the detailed preparation process of HPV16 standards, please refer to the document "Establishment of Quality Control Materials for Human Papillomavirus Genotyping". The general process is as follows: Design the upstream and downstream primers for amplifying the HPV16L1 target region. The primer sequences are: HPV16F (5’-3’), ATGCAGGTGACTTTTATTTACATC; HPV16R (5’-3’), TTACAGCTTACGTTTTTGCGTTT. A fragment of approximately 2000 bp in the L1 region of HPV16 virus in cervical exfoliated cell specimens was amplified by PCR (after sequencing, this fragment was the target sequence of the above primer pair in the HPV16 full genome; the HPV16 full genome sequence is recorded at https:// pave.niaid.nih.gov/database, whose ID is gi|333031|lcl|HPV16REF), and then insert it into the pMD18-T plasmid to construct the recombinant plasmid.
HPV18标准品的详细制备过程可参考文献“人乳头瘤病毒基因分型质控品的建立”。大致过程如下：设计HPV18L1目标区域扩增的上下游引物，引物序列分别为：HPV18F(5’-3’)，ATGTGCCTGTATACA；HPV18R(5’-3’)，TTACTTCCTTGGCACGTACAC。通过PCR扩增宫颈脱落细胞标本中HPV18病毒L1区域中的一个长度约为2000bp的片段(经测序，该片段为HPV18全基因组中上述引物对的靶序列；HPV18全基因组序列记载于https://pave.niaid.nih.gov/数据库，其ID为gi|60975|lcl|HPV18REF)，然后将其插入pMD18-T质粒构建重组质粒。For the detailed preparation process of HPV18 standards, please refer to the document "Establishment of Quality Control Materials for Human Papillomavirus Genotyping". The general process is as follows: Design the upstream and downstream primers for amplification of the HPV18L1 target region. The primer sequences are: HPV18F (5’-3’), ATGTGCCTGTATACA; HPV18R (5’-3’), TTACTTCCTTGGCACGTACAC. A fragment of approximately 2000 bp in the L1 region of the HPV18 virus in cervical exfoliated cell specimens was amplified by PCR (after sequencing, this fragment was the target sequence of the above primer pair in the HPV18 full genome; the HPV18 full genome sequence is recorded at https:// pave.niaid.nih.gov/database, whose ID is gi|60975|lcl|HPV18REF), and then insert it into the pMD18-T plasmid to construct a recombinant plasmid.
供试标准品为HPV16标准品和HPV18标准品。The test standards are HPV16 standard and HPV18 standard.
采用TE缓冲液分别稀释供试标准品，得到标准品稀释液。标准品稀释液中，靶标浓度分别为20000拷贝/μl、2000拷贝/μl或200拷贝/μl。随后将等拷贝数的HPV16标准品稀释液以及HPV18标准品稀释液等体积混合，得到混合标准品稀释液。混合标准品稀释液1中，HPV16靶标浓度和HPV18靶标浓度均为10000拷贝/μl。混合标准品稀释液2中，HPV16靶标浓度和HPV18靶标浓度均为1000拷贝/μl。混合标准品稀释液3中，HPV16靶标浓度和HPV18靶标浓度均为100拷贝/μl。Use TE buffer to dilute the test standards respectively to obtain standard dilutions. In the standard dilution solution, the target concentrations are 20,000 copies/μl, 2,000 copies/μl, or 200 copies/μl. Then, equal volumes of HPV16 standard diluent and HPV18 standard diluent of equal copy numbers were mixed to obtain a mixed standard diluent. In mixed standard dilution 1, the HPV16 target concentration and HPV18 target concentration are both 10,000 copies/μl. In mixed standard dilution 2, the HPV16 target concentration and HPV18 target concentration are both 1000 copies/μl. In mixed standard dilution 3, the HPV16 target concentration and HPV18 target concentration are both 100 copies/μl.
将混合标准品稀释液作为模板，按照实施例2中的步骤一操作，得到50微升PCR产物。设置用等体积TE缓冲液代替混合标准品稀释液，作为空白对照。Use the mixed standard dilution as a template and operate according to step 1 in Example 2 to obtain 50 μl of PCR product. Set up an equal volume of TE buffer instead of the mixed standard diluent as a blank control.
按照实施例2的步骤二制备HPV16-P数码磁珠探针溶液和HPV18-P数码磁珠探针溶液。Prepare HPV16-P digital magnetic bead probe solution and HPV18-P digital magnetic bead probe solution according to step 2 of Example 2.
按照实施例2中的步骤三操作。由于PCR产物的加入量为10微升，因此其对应的模板量为2000、200、20或0拷贝。Follow step three in Example 2. Since the amount of PCR product added is 10 microliters, the corresponding template amount is 2000, 200, 20 or 0 copies.
针对HPV16组，采用HPV16探针所捕获荧光值作为信号值，HPV18探针捕获荧光值作为背景荧光(噪音)值。针对HPV18组，采用HPV18探针所捕获荧光值作为信号值，HPV16探针捕获荧光值作为背景荧光(噪音)值。For the HPV16 group, the fluorescence value captured by the HPV16 probe was used as the signal value, and the fluorescence value captured by the HPV18 probe was used as the background fluorescence (noise) value. For the HPV18 group, the fluorescence value captured by the HPV18 probe was used as the signal value, and the fluorescence value captured by the HPV16 probe was used as the background fluorescence (noise) value.
信号噪音比＝信号值/噪音值。Signal-to-noise ratio = signal value/noise value.
结果见图3。图3中：HPV16对应的4个柱形，自左至右依次对应混合标准品稀释液1、混合标准品稀释液2、混合标准品稀释液3和空白对照；HPV18对应的4个柱形，自左至右依次对应混合标准品稀释液1、混合标准品稀释液2、混合标准品稀释液3和空白对照。信噪比均高于10，该技术方案可以有效的进行HPV核酸的联合检测，针对HPV16/HPV18的检测灵敏度为20拷贝每微升。The results are shown in Figure 3. In Figure 3: the 4 columns corresponding to HPV16, from left to right, correspond to mixed standard dilution 1, mixed standard diluent 2, mixed standard diluent 3 and blank control; the 4 columns corresponding to HPV18, From left to right, they correspond to mixed standard diluent 1, mixed standard diluent 2, mixed standard diluent 3 and blank control. The signal-to-noise ratios are both higher than 10. This technical solution can effectively perform joint detection of HPV nucleic acids, and the detection sensitivity for HPV16/HPV18 is 20 copies per microliter.
工业应用Industrial applications
本发明公开了一种通用的利用数码磁珠实现不同DNA靶标的联合检测方法，核心思想是通过特异性的靶向扩增引物以及相应捕获探针实现靶标的检测。示例性的，本方案发明中只将该技术用于HPV不同亚型的检测实践。事实上，任何核酸靶标都可以利用该方案通过设计特异性的引物与探针实现联合检测，如例如可以利用该技术实现多种呼吸道感染源、肠胃炎感染源等的甄别实践。The invention discloses a universal joint detection method of different DNA targets using digital magnetic beads. The core idea is to realize target detection through specific targeted amplification primers and corresponding capture probes. Illustratively, in this invention, this technology is only used for the detection of different subtypes of HPV. In fact, any nucleic acid target can be jointly detected using this solution by designing specific primers and probes. For example, this technology can be used to screen multiple sources of respiratory tract infections, gastroenteritis infections, etc.

Claims (19)

  1. 一种用于检测n种目标核酸的方法，包括如下步骤：A method for detecting n target nucleic acids, including the following steps:
    (1)将待测核酸进行扩增反应；所述扩增反应的目的为：当待测核酸中含有目标核酸时，通过扩增反应获得末端修饰有基团甲的扩增产物；(1) The nucleic acid to be tested is subjected to an amplification reaction; the purpose of the amplification reaction is: when the nucleic acid to be tested contains a target nucleic acid, an amplification product with a terminal modified with group A is obtained through the amplification reaction;
    (2)将所述扩增反应的产物与n种偶联探针的数码磁珠进行杂交；每种偶联探针的数码磁珠可以结合一种目标核酸分子；偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将一种用于捕获目标核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；(2) Hybridize the product of the amplification reaction with n digital magnetic beads coupled to probes; each digital magnetic bead coupled to the probe can bind to one target nucleic acid molecule; the digital magnetic beads coupled to the probe Beads are obtained by coupling a probe used to capture target nucleic acids and digital magnetic beads; each digital magnetic bead coupled to a probe is obtained by coupling a probe used to capture target nucleic acids and a digital magnetic bead. Obtained; each type of digital magnetic beads has the same barcode, and different types of digital magnetic beads have different barcodes;
    (3)将所述杂交的产物与检测元件进行反应；检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；(3) React the hybridized product with a detection element; the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
    (4)将所述反应后的体系进行如下检测：检测磁珠的条形码以及光信号，根据条形码和光信号确定待测核酸中是否含有目标核酸和/或确定待测核酸中含有n种目标核酸中的哪一种或者哪几种；(4) The reaction system is tested as follows: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains the target nucleic acid and/or determine whether the nucleic acid to be tested contains n kinds of target nucleic acids based on the barcode and light signal. Which type or types;
    n为2以上的自然数。n is a natural number above 2.
  2. 如权利要求1所述的方法，其特征在于：步骤(1)中，所述扩增反应为非对称PCR扩增反应。The method of claim 1, wherein in step (1), the amplification reaction is an asymmetric PCR amplification reaction.
  3. 如权利要求2所述的方法，其特征在于：所述非对称PCR扩增反应，扩增体系中上游引物和下游引物的浓度不同。The method of claim 2, characterized in that: in the asymmetric PCR amplification reaction, the concentrations of upstream primers and downstream primers in the amplification system are different.
  4. 如权利要求1至3中任一所述的方法，其特征在于：所述步骤(1)中，所述扩增反应采用的引物对由上游引物和下游引物组成；下游引物的5'末端修饰有所述基团甲。The method according to any one of claims 1 to 3, characterized in that: in the step (1), the primer pair used in the amplification reaction consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified There is the group A.
  5. 如权利要求1至3中任一所述的方法，其特征在于：所述基团甲为生物素；所述基团乙为链霉亲和素；所述光信号为荧光信号。The method according to any one of claims 1 to 3, characterized in that: the group A is biotin; the group B is streptavidin; and the light signal is a fluorescence signal.
  6. 如权利要求1至3中任一所述的方法，其特征在于：所述探针的5'末端具NH 2C 12修饰。 The method according to any one of claims 1 to 3, characterized in that the 5' end of the probe is modified with NH 2 C 12 .
  7. 一种用于检测n种目标核酸的试剂盒，包括由n个引物对组成的扩增引物组、由n种偶联探针的数码磁珠组成的数码磁珠组和检测元件；A kit for detecting n kinds of target nucleic acids, including an amplification primer set composed of n primer pairs, a digital magnetic bead set composed of n kinds of digital magnetic beads coupled to probes, and a detection element;
    扩增引物组中：不同引物对用于扩增不同的目标核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；In the amplification primer set: different primer pairs are used to amplify different target nucleic acids; each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
    数码磁珠组中：偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将一种用于捕获目标核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；In the digital magnetic bead set: the digital magnetic beads for coupling probes are obtained by coupling the probe used to capture the target nucleic acid with the digital magnetic beads; each type of digital magnetic beads for coupling probes is obtained by coupling one type of digital magnetic beads for capturing target nucleic acids. The target nucleic acid probe is coupled to a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcodes;
    检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；The detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
    n为2以上的自然数。n is a natural number above 2.
  8. 如权利要求7所述的试剂盒，其特征在于：所述基团甲为生物素；所述基团乙为链霉亲和素；所述光信号为荧光信号。The kit according to claim 7, characterized in that: the group A is biotin; the group B is streptavidin; and the light signal is a fluorescence signal.
  9. 如权利要求7或8所述的试剂盒，其特征在于：所述探针的5'末端具NH 2C 12修饰。 The kit according to claim 7 or 8, wherein the 5' end of the probe is modified with NH 2 C 12 .
  10. 一种用于检测HPV的方法，包括如下步骤：A method for detecting HPV, including the following steps:
    (1)以待测核酸样本为模板，采用扩增引物组进行PCR扩增；扩增引物组由n个引物对组成；每个引物对用于扩增一种型别的HPV的特征性核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；(1) Use the nucleic acid sample to be tested as a template and use an amplification primer set to perform PCR amplification; the amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV ;Each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with a group A;
    (2)将所述PCR扩增的产物与数码磁珠组进行杂交；数码磁珠组由n种偶联探针的数码磁珠组成；偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将用于捕获一种型别的HPV的特征性核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；(2) Hybridize the PCR amplified product with a digital magnetic bead set; the digital magnetic bead set consists of n digital magnetic beads coupled to probes; the digital magnetic beads coupled to probes will be used to capture the target Nucleic acid probes are obtained by coupling to digital magnetic beads; each digital magnetic bead coupling probe is obtained by coupling a probe for capturing the characteristic nucleic acid of a type of HPV to a digital magnetic bead. ; Each type of digital magnetic beads has the same barcode, and different types of digital magnetic beads have different barcodes;
    (3)将所述杂交的产物与检测元件进行反应；检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；(3) React the hybridized product with a detection element; the detection element has group B and group C; the group B specifically binds to the group A; the group C has the ability to emit light The function of the optical signal for detection;
    (4)将所述反应后的体系进行如下检测：检测磁珠的条形码以及光信号，根据条形码和光信号确定待测核酸中是否含有HPV和/或确定待测核酸中含有n种HPV中的哪一种或者哪几种；(4) The reaction system is subjected to the following detection: detect the barcode and light signal of the magnetic beads, determine whether the nucleic acid to be tested contains HPV and/or determine which of the n types of HPV is contained in the nucleic acid to be tested based on the barcode and light signal. One or several types;
    n为2以上的自然数。n is a natural number above 2.
  11. 如权利要求10所述的方法，其特征在于：步骤(1)中，所述PCR扩 增为非对称PCR扩增。The method of claim 10, wherein in step (1), the PCR amplification is asymmetric PCR amplification.
  12. 如权利要求11所述的方法，其特征在于：所述非对称PCR扩增，扩增体系中上游引物和下游引物的浓度不同。The method according to claim 11, characterized in that: in the asymmetric PCR amplification, the concentrations of upstream primers and downstream primers in the amplification system are different.
  13. 如权利要求10至12中任一所述的方法，其特征在于：所述基团甲为生物素；所述基团乙为链霉亲和素；所述光信号为荧光信号。The method according to any one of claims 10 to 12, characterized in that: the group A is biotin; the group B is streptavidin; and the light signal is a fluorescence signal.
  14. 如权利要求10至12中任一所述的方法，其特征在于：所述探针的5'末端具NH 2C 12修饰。 The method according to any one of claims 10 to 12, characterized in that: the 5' end of the probe is modified with NH 2 C 12 .
  15. 如权利要求10至12中任一所述的方法，其特征在于：The method according to any one of claims 10 to 12, characterized in that:
    n为2；n is 2;
    扩增引物组由2个引物对组成；一个引物对用于扩增HPV16的特征性核酸，由HPV16-F和HPV16-R组成；另一个引物对用于扩增HPV18的特征性核酸，由HPV18-F和HPV18-R组成；The amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV18, consisting of HPV18 -F and HPV18-R;
    数码磁珠组由2种偶联探针的数码磁珠组成；一种偶联探针的数码磁珠是将用于捕获HPV16的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV16的特征性核酸的探针命名为HPV16-P；另一种偶联探针的数码磁珠是将用于捕获HPV18的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV18的特征性核酸的探针命名为HPV18-P；The digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is obtained by coupling a probe used to capture the characteristic nucleic acid of HPV16 with a digital magnetic bead. , the probe used to capture the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupling probe is to couple the probe used to capture the characteristic nucleic acid of HPV18 with a digital magnetic bead The obtained probe used to capture the characteristic nucleic acid of HPV18 was named HPV18-P;
    HPV16-F的核苷酸序列如SEQ ID NO：1所示；HPV16-R的核苷酸序列如SEQ ID NO：2所示；HPV16-P的核苷酸序列如SEQ ID NO：3所示；HPV18-F的核苷酸序列如SEQ ID NO：4所示；HPV18-R的核苷酸序列如SEQ ID NO：5所示；HPV18-P的核苷酸序列如SEQ ID NO：6所示。The nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleotide sequence of HPV16-P is shown in SEQ ID NO: 3 ; The nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleotide sequence of HPV18-P is shown in SEQ ID NO: 6 Show.
  16. 一种用于检测HPV的试剂盒，包括扩增引物组、数码磁珠组和检测元件；A kit for detecting HPV, including an amplification primer set, a digital magnetic bead set and a detection element;
    扩增引物组由n个引物对组成；每个引物对用于扩增一种型别的HPV的特征性核酸；每个引物对由上游引物和下游引物组成；下游引物的5'末端修饰有基团甲；The amplification primer set consists of n primer pairs; each primer pair is used to amplify the characteristic nucleic acid of one type of HPV; each primer pair consists of an upstream primer and a downstream primer; the 5' end of the downstream primer is modified with Group A;
    数码磁珠组由n种偶联探针的数码磁珠组成；偶联探针的数码磁珠是将用于捕获目标核酸的探针和数码磁珠偶联得到的；每种偶联探针的数码磁珠是将用于捕获一种型别的HPV的特征性核酸的探针和一种数码磁珠偶联得到的；每种数码磁珠具有相同的条形码，不同种数码磁珠具有不同的条形码；The digital magnetic bead set consists of n types of digital magnetic beads coupled to probes; the digital magnetic beads coupled to probes are obtained by coupling the probe used to capture the target nucleic acid and the digital magnetic beads; each coupled probe The digital magnetic beads are obtained by coupling a probe used to capture the characteristic nucleic acid of a type of HPV with a digital magnetic bead; each digital magnetic bead has the same barcode, and different types of digital magnetic beads have different barcode;
    检测元件中具有基团乙和基团丙；所述基团乙与所述基团甲特异性结合；所述基团丙具有发出可供检测的光信号的功能；The detection element has group B and group C; the group B specifically binds to the group A; the group C has the function of emitting a light signal for detection;
    n为2以上的自然数。n is a natural number above 2.
  17. 如权利要求16所述的试剂盒，其特征在于：所述基团甲为生物素；所述基团乙为链霉亲和素；所述光信号为荧光信号。The kit according to claim 16, characterized in that: the group A is biotin; the group B is streptavidin; and the light signal is a fluorescence signal.
  18. 如权利要求16所述的试剂盒，其特征在于：所述探针的5'末端具NH 2C 12修饰。 The kit of claim 16, wherein the 5' end of the probe is modified with NH 2 C 12 .
  19. 如权利要求16或17或18所述的试剂盒，其特征在于：The kit according to claim 16, 17 or 18, characterized in that:
    n为2；n is 2;
    扩增引物组由2个引物对组成；一个引物对用于扩增HPV16的特征性核酸，由HPV16-F和HPV16-R组成；另一个引物对用于扩增HPV18的特征性核酸，由HPV18-F和HPV18-R组成；The amplification primer set consists of 2 primer pairs; one primer pair is used to amplify the characteristic nucleic acid of HPV16, consisting of HPV16-F and HPV16-R; the other primer pair is used to amplify the characteristic nucleic acid of HPV18, consisting of HPV18 -F and HPV18-R;
    数码磁珠组由2种偶联探针的数码磁珠组成；一种偶联探针的数码磁珠是将用于捕获HPV16的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV16的特征性核酸的探针命名为HPV16-P；另一种偶联探针的数码磁珠是将用于捕获HPV18的特征性核酸的探针和一种数码磁珠偶联得到的，用于捕获HPV18的特征性核酸的探针命名为HPV18-P；The digital magnetic bead set consists of two types of digital magnetic beads coupled to probes; one type of digital magnetic beads coupled to probes is obtained by coupling a probe used to capture the characteristic nucleic acid of HPV16 with a digital magnetic bead. , the probe used to capture the characteristic nucleic acid of HPV16 is named HPV16-P; another digital magnetic bead coupling probe is to couple the probe used to capture the characteristic nucleic acid of HPV18 with a digital magnetic bead The obtained probe used to capture the characteristic nucleic acid of HPV18 was named HPV18-P;
    HPV16-F的核苷酸序列如SEQ ID NO：1所示；HPV16-R的核苷酸序列如SEQ ID NO：2所示；HPV16-P的核苷酸序列如SEQ ID NO：3所示；HPV18-F的核苷酸序列如SEQ ID NO：4所示；HPV18-R的核苷酸序列如SEQ ID NO：5所示；HPV18-P的核苷酸序列如SEQ ID NO：6所示。The nucleotide sequence of HPV16-F is shown in SEQ ID NO: 1; the nucleotide sequence of HPV16-R is shown in SEQ ID NO: 2; the nucleotide sequence of HPV16-P is shown in SEQ ID NO: 3 ; The nucleotide sequence of HPV18-F is shown in SEQ ID NO: 4; the nucleotide sequence of HPV18-R is shown in SEQ ID NO: 5; the nucleotide sequence of HPV18-P is shown in SEQ ID NO: 6 Show.
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