WO2022033334A1 - 一种新型冠状病毒(SARS-CoV-2)快速检测试剂盒及其方法 - Google Patents

一种新型冠状病毒(SARS-CoV-2)快速检测试剂盒及其方法 Download PDF

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WO2022033334A1
WO2022033334A1 PCT/CN2021/109788 CN2021109788W WO2022033334A1 WO 2022033334 A1 WO2022033334 A1 WO 2022033334A1 CN 2021109788 W CN2021109788 W CN 2021109788W WO 2022033334 A1 WO2022033334 A1 WO 2022033334A1
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exonuclease
modification
novel coronavirus
amplification
rapid detection
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French (fr)
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于文强
徐鹏
董世华
朱同玉
徐建青
茹道平
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上海奕谱生物科技有限公司
上海市公共卫生临床中心
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    • 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
    • C12Q1/701—Specific hybridization probes
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of biotechnology, in particular to a novel coronavirus rapid detection kit and method thereof.
  • SARS-CoV-2 The pneumonia epidemic (COVID-19) caused by the new coronavirus (SARS-CoV-2) has caused great harm to the world. So far, after more than 7 months of transmission, SARS has spread all over the world. -CoV-2 still has no trend of decay. The current detection of SARS-CoV-2 mainly relies on public health systems such as hospitals and professional medical staff, which is time-consuming, inefficient, and has a high risk factor.
  • SARS-CoV-2 is a class of positive-sense single-stranded RNA viruses.
  • the current detection strategies are mainly divided into two types: 1. Detection based on IgG and IgM antibodies. This detection method is suitable for a period of time after infection, but is not suitable for early infection or asymptomatic infection, and has obvious limitations; 2. , Nucleic acid-based detection, which is the current "gold standard”. In terms of nucleic acid detection, there are mainly three types: (1) conventional real-time quantitative PCR detection, which is the most common method at present. One or more pairs of primers are designed for the new coronavirus sequence, nucleic acid is extracted, and reverse transcription is performed.
  • the present invention overcomes the defects of the prior art, and provides a rapid and accurate detection kit and method for the novel coronavirus.
  • a first aspect of the present invention provides a novel coronavirus (SARS-CoV-2) rapid detection kit and method thereof, comprising the following steps:
  • step S1 Obtain the sample to be detected, use the magnetic bead adsorption method to obtain the components to be detected in the sample, and lyse the adsorbed cells or pathogens by the lysis method to release the nucleic acids in the cells or pathogens; step S1 can be replaced with any other The way to obtain sample nucleic acid, such as phenol chloroform extraction, adsorption column extraction, etc.;
  • step S2 Perform multiple isothermal amplifications on the nucleic acid samples obtained in step S1, and the last isothermal amplification is isothermal amplification for generating nucleic acid single strands; step S2 can be replaced with any other method capable of generating nucleic acid single strands, such as T7 nucleic acid Exonuclease treatment, in vitro transcription, etc.;
  • step S3 Hybridize the single-stranded product obtained by multiple isothermal amplifications or other methods in step S2 with the modified probe, and the probe types include DNA probes, RNA probes, LNA-modified probes, and peptide nucleic acid probes Wait;
  • step S4 adding a specific exonuclease to the hybridized product in step S3 to excise excess probes;
  • step S2 further includes: adding reverse transcriptase to the RNA nucleic acid sample obtained in S1 to reverse-transcribe the RNA nucleic acid sample into a DNA nucleic acid sample.
  • the samples in step S1 include body fluids, mouthwashes, saliva, nasal and throat swab samples, sputum, scrapes, urine, feces, body secretions, and tissue samples; further preferred , the sample types are mouthwash, saliva, nasal swab specimen and throat swab specimen.
  • the magnetic beads used in the magnetic bead adsorption method in step S1 include concanavalin A-coated magnetic beads.
  • the probe in step S3 is a modified oligonucleotide probe; further preferably, the types of probe modifications include: biotin modification (Biotin), digoxigenin modification (Digoxigenin), isothiocyanate Acid fluorescein modification (FITC), Texas red modification (Texas Red-X), phosphorylation modification (Phosphorylation), amino modification (Aminolinker C6/7/12), thiol modification (Thiol C6/Thiol-C6 SS) , Phosphorothioate, fluorescent group modification (FAM/HEX/ROX/CY-3/CY-5/JOE/TET, etc.), quenching group modification (MGB/BHQ1/BHQ2/TAMRA, etc.); more Further preferably, the probe modification is: digoxigenin (DIGOXIGENIN) modification, biotin (BIOTIN) modification.
  • biotin modification Digoxigenin
  • FITC isothiocyanate Acid fluorescein modification
  • the base sequence of the probe is SEQ ID NO.1.
  • SEQ ID NO. 1 CAGCCATAACCTTTCCACATACCGCAGACG.
  • the specific exonuclease in step S4 includes, exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III , RecJ1 exonuclease, exonuclease I; more preferably, the exonuclease is exonuclease VII.
  • the lysis method described in step S1 includes using a lysis solution for cell lysis.
  • the lysate comprises 10 mM HEPES, 0.3% SDS, 0.5% IGEPAL-CA630, 1.5 mM MgCl2, 0.1% diethylpyrocarbonate, 10 mM KCl, 50 mM Tris, 10 mM EDTA, 1% RNase inhibitor, and 1% protease in water.
  • the isothermal amplification described in step S2 includes recombinase polymerase amplification technology, recombinase-assisted amplification technology, loop-mediated isothermal amplification technology, helicase-dependent isothermal amplification technology, strand displacement technology isothermal amplification technology, nucleic acid sequence-based isothermal amplification technology, rolling circle amplification technology, and nickase amplification technology.
  • Recombinase Polymerase Amplification It mainly relies on the recombinase, single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase that can bind to single-stranded nucleic acid (oligonucleotide primer). It is a protein-DNA complex formed by the combination of recombinase and primer, which can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequence, a strand-exchange reaction takes place and initiates DNA synthesis, exponentially amplifying the target region on the template. The replaced DNA strand binds to the SSB, preventing further replacement. In this system, a synthesis event is initiated by two opposing primers. The whole process can be carried out at room temperature, and generally a detectable level of amplification product can be obtained in about 10 minutes.
  • Recombinase-aided amplification It is a method that can rapidly amplify nucleic acid at room temperature.
  • RAA uses recombinase obtained from bacteria or fungi. The recombinase can be tightly combined with the primer to form a polymer.
  • the primer is paired with the template, with the help of single-stranded DNA binding protein, the template DNA is unchained, and under the action of DNA polymerase, new DNA is formed complementary strands.
  • Loop-mediated isothermal amplification The LAMP method is to design 4-6 primers for the region to be detected on the target gene, and use strand displacement DNA polymerase to amplify under constant temperature conditions The reaction can be amplified within 15-60 minutes under the condition of 60-65°C, and the reaction can produce a large amount of amplification products.
  • Strand displacement isothermal amplification technology The nickase-based strand displacement isothermal amplification technology established by this technology is simpler, faster, more environmentally friendly and less expensive than traditional methods. It has very strong practical application potential and is especially suitable for on-site detection. .
  • Rolling circle amplification uses a circular DNA as a template to synthesize new circular DNA molecules. First, a nick is created at the origin of replication of a single strand, and then new single strands are continuously synthesized using the other single strand as a template. The newly synthesized single-stranded DNA released is first copied into double-stranded DNA, and then cut into a unit length by the enzyme, and then a circular double-stranded DNA molecule is formed; or the newly synthesized single-stranded DNA released is first cut by the enzyme. A single-stranded circular DNA molecule is formed into a unit length and then replicated into a double-stranded circular DNA molecule.
  • Nucleic acid sequence-based amplification is a continuous and uniform process that uses RNA in the nucleic acid sequence as a template and is mediated by two primers. The specificity of an enzymatic process for isothermal amplification of nucleotide sequences in vitro.
  • the whole reaction consists of acyclic phase and cyclic phase: firstly, the acyclic phase is carried out.
  • primer 1 and template RNA are annealed to synthesize cDNA to form RNA/DNA hybrid, and then RNaseH degrades RNA, primer 2 Annealed with cDNA to synthesize a second complementary DNA strand.
  • the formed DNA duplex is recognized by the T7 RNA polymerase promoter sequence, catalyzes the synthesis of RNA, enters the circulation phase, and amplifies the template in large quantities.
  • HSA Helicase-dependent isothermal DNA amplification
  • NEAR is a strand displacement amplification technology. The raw material is polymerized and extended from the 3' end of the cleft to replace the allelic DNA strand, thereby forming a new and complete DNA sequence containing a nickase recognition site. This double-strand is recognized and cut by nucleic acid nicking endonuclease again, and then starts the cycle of "polymerization-nicking", resulting in a large number of displaced DNA single-strands, forming exponential amplification.
  • the multiple isothermal amplifications described in step S2 include sequentially performing one recombinase polymerase amplification and one nicking endonuclease amplification.
  • the nickases used in the nickase amplification are Nb.BtsI, Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.
  • the strand displacement nucleic acid polymerase used in the nickase amplification is Bst3.0 DNA polymerase, Bst2.0 DNA polymerase, Klenow DNA polymerase, phi29 DNA polymerase; further preferably, the nickase used for the nickase amplification is Nb.BtsI, and the nickase amplification adopts the strand displacement nucleic acid polymerase It is Bst3.0 DNA polymerase.
  • the basic sequence of the forward primer includes the nicking site CACTGC of the nicking endonuclease and the protective base, and the sequence of the forward primer is: SEQ ID NO.2; the sequence of the reverse primer is : SEQ ID NO.3.
  • the protected base is in the box
  • the single thick underline is the nicking site
  • the double thin underline is the template binding sequence.
  • SEQ ID NO. 3 CCCGTTTAAAAACGATTGTGCATCAGCTGAC.
  • the method for detecting hybridization products in step S5 includes colloidal gold method and colloidal carbon method; further preferably, the method for detecting hybridization products is colloidal gold test strips.
  • the second aspect of the present invention provides a novel coronavirus rapid detection kit, including magnetic beads, lysis solution, isothermal amplification primers, isothermal amplification enzymes, probes, specific exonuclease, and detection test paper.
  • the magnetic beads comprise concanavalin A-coated magnetic beads.
  • the probe contains biotin and digoxigenin modification; further preferably, the base sequence of the probe is SEQ ID NO.1
  • the specific exonuclease includes, exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III, RecJ1 nucleic acid Exonuclease, exonuclease I; more preferably, the exonuclease is exonuclease VII.
  • the lysate comprises 10 mM HEPES, 0.3% SDS, 0.5% IGEPAL-CA630, 1.5 mM MgCl2, 0.1% diethylpyrocarbonate, 10 mM KCl, 50 mM Tris, 10 mM EDTA, 1% RNase inhibitor, and 1% protease in water.
  • the isothermal amplification enzyme includes a recombinase polymerase and a nickase; further preferably, the basic sequence of the isothermal amplification forward primer includes the nicking sites of the nickase CACTGC and Protect the base, the forward primer sequence is: SEQ ID NO.2; the reverse primer sequence is: SEQ ID NO.3.
  • the detection test paper includes a colloidal gold test paper strip.
  • the third aspect of the present invention provides the application of the kit of the present invention in the preparation of reagents for detecting novel coronaviruses and/or detecting diseases caused by novel coronaviruses.
  • the disease caused by the novel coronavirus includes novel coronavirus pneumonia.
  • the present invention Compared with the prior art, the present invention has the following advantages: the present invention develops a set of SARS-CoV-2 detection scheme that can be checked at home. In the early stage, the SARS-CoV-2 standard DNA and RNA were tested, with high sensitivity and specificity; the nucleic acid extracted from the nasal swabs and throat swabs of the new crown patients was tested, and 4 cases were detected in 5 cases. And samples with a cycle number of 37 can be detected, indicating that this method has extremely high sensitivity.
  • Fig. 1 is the nucleic acid verification result of the cells in the mouthwash based on magnetic bead enrichment and lysis solution lysis in Example 1 of the present invention
  • Fig. 2 is the result of cleavage of the probe by the probe and single-stranded exonuclease in Example 2 of the present invention
  • Fig. 3 is the detection result of using novel coronavirus DNA and RNA standard substance in the embodiment of the present invention 2;
  • FIG. 4 is the detection result of nucleic acid purified from the nasal swab or throat swab of the new crown patient in Example 3 of the present invention.
  • FIG. 5 is the test result of using the new crown patient mouthwash in Example 4 of the present invention.
  • Example 1 Using magnetic beads and lysate to release nucleic acid in cells and carry out PCR verification
  • the PCR template of the negative control is RPMI1640 medium
  • the PCR template of the positive control 1 is HEK293 genomic DNA
  • the PCR template of the positive control 2 is obtained by lysing oral epithelial cells using a commercial kit cell nucleic acid
  • the PCR template of the experimental group was magnetic beads and lysate to release cell nucleic acid.
  • the base sequence of ⁇ -actin-F primer is: SEQ ID NO.4; the base sequence of ⁇ -actin-R primer is SEQ ID NO.5.
  • SEQ ID NO. 5 AGCACTGTGTTGGCGTACAG
  • the nucleic acid sequence of the probe is: SEQ ID NO.1, wherein the 5' end is labeled with digoxigenin modification, and the 3' end is labeled with biotin modification.
  • the negative control group (sample 1) is water, and the experimental groups are: 1 pmol of the modified probe (sample 2), 10 pmol of the modified probe (sample 3), and 10 pmol of the modified probe covered by single-stranded After digestion with exonuclease Exo VII (sample 4).
  • Example 3 Detection of new coronavirus (SARS-CoV-2) DNA standards and RNA standards 3.1. Preparation of test samples: the negative control group (sample 1) used water, and the experimental group used 1000 copies of SARS-CoV-2 DNA fragment (sample 2), 10,000 copies of SARS-CoV-2 RNA standard (sample 3), and 1,000 copies of SARS-CoV-2 RNA standard (sample 4).
  • the negative control group used water
  • the experimental group used 1000 copies of SARS-CoV-2 DNA fragment (sample 2), 10,000 copies of SARS-CoV-2 RNA standard (sample 3), and 1,000 copies of SARS-CoV-2 RNA standard (sample 4).
  • the first isothermal amplification Take the detection sample obtained in step 1 as a template, and use the RNA isothermal rapid amplification kit (Amp Future, WLRN8206KIT) to carry out isothermal amplification. Refer to the kit instructions for isothermal amplification steps.
  • the reaction system is as follows:
  • the forward reactant F is: SEQ ID NO.2;
  • Reverse primer R is: SEQ ID NO.3.
  • Secondary amplification add 1 ⁇ l Nb.BtsI, 1 ⁇ l Bst3.0, 4 ⁇ l dNTPs, react at 37°C for 15 minutes, and 95°C for 5 minutes.
  • Probe hybridization add 2 ⁇ l of 10 ⁇ M labeled probe (detection probe provided in Example 2), carry out hybridization at 95° C. for 1 min, and then slowly reduce to room temperature (about 15 min).
  • Colloidal gold test strip detection add 60 ⁇ l ddH2O, mix well, insert the colloidal gold test strip into the liquid, and read the result within 5 minutes.
  • the nucleic acid in the throat swab sample was extracted by the method provided in Example 1.
  • Example 3 Water was used as the negative control group, 1000 copies of SARS-CoV-2 DNA fragments were used as the positive control group, and the nucleic acids of 5 patients obtained in step 4.1 were used as the experimental group, and the method of Example 3 was used for detection.
  • Example 2 Use the method provided in Example 1 to extract the nucleic acid in the mouthwash sample.
  • Example 3 Water was used as the negative control group, 1000 copies of SARS-CoV-2 DNA fragments were used as the positive control group, and the nucleic acids of 4 patients obtained in step 5.1 were used as the experimental group, and the method of Example 3 was used for detection.

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Abstract

本发明提供一种新型冠状病毒快速检测方法，包括以下步骤：S1：获取待测样本；S2：将核酸样本进行多次恒温扩增，且最后一次恒温扩增为产生核酸单链的恒温扩增，或产生单链的其他方法；S3：将步骤S2多次恒温扩增或其他方法得到的单链产物与修饰的探针进行杂交；S4：将步骤S3杂交后的产物中加入特异性外切酶，切除多余的探针；S5：检测步骤S4的产物。本发明还提供一种新型冠状病毒快速检测试剂盒，包括磁珠、细胞裂解液、恒温扩增引物、恒温扩增酶、探针、特异性外切酶、检测试纸。

Description

一种新型冠状病毒(SARS-CoV-2)快速检测试剂盒及其方法 技术领域
本发明涉及生物技术领域，尤其涉及一种新型冠状病毒快速检测试剂盒及其方法。
背景技术
新型冠状病毒(SARS-CoV-2)引发的肺炎疫情(COVID-19)给全世界造成了极大的危害，到目前为止，已经经历了超过7个月的传播后，在全世界范围内SARS-CoV-2依然没有衰减的趋势。目前的SARS-CoV-2的检测主要依赖于医院等公共卫生系统及专业的医护人员，耗时长，效率低，危险系数高。
SARS-CoV-2是一类正义单链RNA病毒。目前检测策略主要分为两种：1、基于IgG和IgM抗体的检测，该检测方法适用于感染一段时间后，对感染初期或无症感染者的适用性不高，存在明显的局限性；2、基于核酸的检测，这是目前的“金标准”。在核酸检测方面，主要分为三种：(1)常规的实时定量荧光PCR检测，这是目前最通用的方法，针对新冠病毒序列设计一对或多对引物，抽提核酸，进行反转录并进行qPCR，检测结果为循环数；(2)不依赖大型仪器的现场检测方法，比如美国研究者开发了基于CRISPR/Cas12的方法DETECTER，该方法采用的样本是鼻拭子或咽拭子中提取的核酸，无法满足病人自取样和自操作的需求；(3)居家快速检测，即检测者可自取样、自操作、自读取结果的简单的检测方案。
居家型检测不依赖于大型仪器，可自己取样的新冠病毒快速检测方法，将极大提高SARS-CoV-2的检测效率和安全性，将为疫情的防控和国家政策的制定提供了强有力的手段。但是，现有技术还缺少一种针对新型冠状病毒快速、准确的进行快速检测的试剂盒及其方法。
发明内容
本发明克服现有技术的缺陷，提供了一种针对新型冠状病毒快速、准确的进行检测的试剂盒及其方法。
本发明的第一个方面，提供了一种新型冠状病毒(SARS-CoV-2)快速检测试剂盒及其方法，包括以下步骤：
S1：获取待检测的样本，采用磁珠吸附法获取样本中的待检测组分，并通过裂解法将吸附的细胞或病原体裂解，释放出细胞或病原体内的核酸；步骤S1可以替换成其他任何可以获取样本核酸的方式，比如酚氯仿抽提、吸附柱提取等；
S2：将步骤S1得到的核酸样本进行多次恒温扩增，且最后一次恒温扩增为产生核酸单链的恒温扩增；步骤S2可以替换成其他任何能够产生核酸单链的方式，比如T7核酸外切酶处理，体外转录等；
S3：将步骤S2多次恒温扩增或其他方法得到的单链产物与修饰的探针进行杂交，所述探针类型包括DNA探针、RNA探针、LNA修饰的探针、肽核酸探针等；
S4：将步骤S3杂交后的产物中加入特异性外切酶，切除多余的探针；
S5：检测S4的产物。
优选的，步骤S2还包括：向S1得到的RNA核酸样本中加入反转录酶，使RNA核酸样本反转录为DNA核酸样本。
优选的，步骤S1中所述样本包括体液、漱口液、唾液、鼻拭子标本和咽拭子标本、痰液、刮片、尿液、粪便、机体分泌物、组织样本；更进一步优选的，所述样本类型为漱口液、唾液、鼻拭子标本和咽拭子标本。
优选的，步骤S1中所述磁珠吸附法使用的磁珠包括伴刀豆球蛋白A包被磁珠。
优选的，步骤S3中所述探针为修饰寡核苷酸探针；进一步优选的，所述探针修饰的类型包括：生物素修饰(Biotin)、地高辛修饰(Digoxigenin)、异硫氰酸荧光素修饰(FITC)、德克萨斯红修饰(Texas Red-X)、磷酸化修饰(Phosphorylation)、氨基修饰(Aminolinker C6/7/12)、巯基修饰(Thiol C6/Thiol-C6 S-S)、硫代修饰(Phosphorothioate)、荧光基团修饰(FAM/HEX/ROX/CY-3/CY-5/JOE/TET等)、淬灭基团修饰(MGB/BHQ1/BHQ2/TAMRA等)；更进一步优选的，所述探针修饰为：地高辛(DIGOXIGENIN)修饰，生物素(BIOTIN)修饰。
优选的，所述探针的碱基序列为SEQ ID NO.1。
SEQ ID NO.1 CAGCCATAACCTTTCCACATACCGCAGACG。
优选的，步骤S4中所述特异性外切酶包括，核酸外切酶VII、λ核酸外切酶、T5核酸外切酶、T7核酸外切酶、核酸外切酶V、核酸外切酶III、RecJ1核酸外切酶、核酸外切酶I；更进一步优选的，所述外切酶为核酸外切酶VII。
优选的，步骤S1所述的裂解法包括采用裂解液进行细胞裂解。
优选的，所述裂解液包括10mM HEPES,0.3％SDS,0.5％IGEPAL-CA630,1.5mM MgCl2,0.1％焦碳酸二乙酯，10mM KCl，50mM Tris,10mM EDTA,1％RNA酶抑制剂，和1％蛋白酶的水溶液。
优选的，步骤S2中所述的恒温扩增包括重组酶聚合酶扩增技术、重组酶辅助的扩增技术、环介导的等温扩增技术、解旋酶依赖的等温扩增技术、链置换的等温扩增技术、基于核酸序列的等温扩增技术、滚环扩增技术、切刻内切酶扩增技术。
重组酶聚合酶恒温扩增技术(Recombinase Polymerase Amplification，RPA)：主要依赖能结合单链核酸(寡核苷酸引物)的重组酶、单链DNA结合蛋白(SSB)和链置换DNA聚合酶这三种酶，重组酶与引物结合形成的蛋白-DNA复合物，能在双链DNA中寻找同源序列。一旦引物定位了同源序列，就会发生链交换反应形成并启动DNA合成，对模板上的目标区域进行指数式扩增。被替换的DNA链与SSB结合，防止进一步替换。在这个体系中，由两个相对的引物起始一个合成事件。整个过程可在室温进行，一般可在10分钟左右获得可检出水平的扩增产物。
重组酶辅助的扩增技术(recombinase-aided amplification，RAA)：是一种在室温下可以快速扩增核酸的方法，RAA使用的是从细菌或真菌中获得的重组酶，在37℃恒温下，该重组酶可以与引物紧密结合，形成聚合体，当引物与模板进行配对后，在单链DNA结合蛋白的帮助下，使模板DNA解链，并在DNA聚合酶的作用下，形成新的DNA互补链。
环介导的等温扩增技术(Loop-mediated isothermal amplification，LAMP)：LAMP法是针对靶基因上的待检测区域设计4-6条引物，利用链置换型DNA聚合 酶在恒温条件下进行扩增反应，可在60～65℃条件下，15-60分钟内实现扩增，反应能产生大量的扩增产物。
链置换的等温扩增技术：本技术建立的基于缺口酶的链置换等温扩增技术，较之传统方法更加简便、快捷、环保且价格低廉,具有非常强的实际应用潜力,特别适用于现场检测。
滚环扩增：滚环式复制是以一条环状DNA为模板，进行新的DNA环状分子合成。先在一条单链的复制起点上产生一个切口，然后以另一条单链为模板不断地合成新的单链。释放出的新合成的单链或是先复制成双链DNA，被酶切割成单位长度后，再形成环状双链DNA分子；或是释放出的新合成的单链DNA，先被酶切割成单位长度形成单链环状DNA分子后再复制成双链环状DNA分子。
基于核酸序列的等温扩增技术(Nucleic acid sequence-based amplification,NASBA)：依赖核酸序列的扩增技术(NASBA)是一项以核酸序列中RNA为模板，由两个引物介导的、连续均一的特异性体外等温扩增核苷酸序列的酶促过程。整个反应由非循环相和循环相组成:首先进行非循环相，在AMV逆转录酶的作用下，引物1与模板RNA退火后合成cDNA，形成RNA/DNA杂合体，随即RNaseH降解RNA，引物2与cDNA退火，合成第二条DNA互补链。形成的DNA双链由T7 RNA聚合酶识别启动子序列，催化合成RNA，进入循环相，对模板进行大量扩增。
解旋酶依赖的等温扩增技术(Helicase-dependent isothermal DNA amplification,HDA)：该技术模拟体内DNA复制的自然过程，利用解旋酶在恒温下解开DNA双链，再由DNA单链结合蛋白稳定已解开的单链为引物提供模板，然后在DNA聚合酶的作用下合成互补的双链，继而不断重复上述循环扩增过程，最终实现靶序列的指数式增长。
切刻内切酶扩增反应(nicking endonuclease amplification reaction，NEAR):NEAR是一种链置换放大技术，其原理是在核酸切刻内切酶切刻形成的裂口处，通过聚合酶的作用以dNTPs为原料从裂口处的3’端聚合延伸，置换出等位的DNA链，由此又形成了新的完整的含有切刻酶识别位点的DNA序列。这条双链再次被核酸切刻内切酶识别切割，进而开始“聚合-切刻”的循环，产生大量被置换下来的DNA单链，形成指数级扩增。
优选的，步骤S2所述的多次恒温扩增包括依次进行一次重组酶聚合酶扩增和一次切刻内切酶扩增。
优选的，所述切刻内切酶扩增采用的切刻内切酶为Nb.BtsI、Nt.BspQI、Nt.CviPII、Nt.BstNBI、Nb.BsrDI、Nb.BtsI、Nt.AlwI、Nb.BbvCI、Nt.BbvCI、Nb.BsmI、Nb.BssSI、Nt.BsmAI，所述的切刻内切酶扩增采用的链置换核酸聚合酶为Bst3.0 DNA聚合酶、Bst2.0 DNA聚合酶、Klenow DNA聚合酶、phi29 DNA聚合酶；更进一步优选的，所述切刻内切酶扩增采用的切刻内切酶为Nb.BtsI，切刻内切酶扩增采用的链置换核酸聚合酶为Bst3.0 DNA聚合酶。
优选的，多次恒温扩增，正向引物的碱性序列包含切刻内切酶的切刻位点CACTGC和保护碱基，正向引物序列为：SEQ ID NO.2；反向引物序列为：SEQ ID NO.3。
SEQ ID NO.2
Figure PCTCN2021109788-appb-000001
Figure PCTCN2021109788-appb-000002
其中方框中为保护碱基，单粗下划线为切刻位点，双细下划线为模板结合序列。
SEQ ID NO.3 CCCGTTTAAAAACGATTGTGCATCAGCTGAC。
优选的，步骤S5中检测杂交产物的方法包括胶体金法、胶体碳法；进一步优选的，检测杂交产物的方法为胶体金试纸条。
本发明的第二个方面，提供了一种新型冠状病毒快速检测试剂盒，包括磁珠、裂解液、恒温扩增引物、恒温扩增酶、探针、特异性外切酶、检测试纸。
优选的，所述磁珠包括伴刀豆球蛋白A包被磁珠。
优选的，所述探针含有生物素与地高辛修饰；进一步优选的，所述探针的碱基序列为SEQ ID NO.1
优选的，所述特异性外切酶包括，核酸外切酶VII、λ核酸外切酶、T5核酸外切酶、T7核酸外切酶、核酸外切酶V、核酸外切酶III、RecJ1核酸外切酶、核酸外切酶I；更进一步优选的，所述外切酶为核酸外切酶VII。
优选的，所述裂解液包括10mM HEPES,0.3％SDS,0.5％IGEPAL-CA630,1.5mM MgCl2,0.1％焦碳酸二乙酯，10mM KCl，50mM Tris,10mM EDTA,1％RNA酶抑制剂，和1％蛋白酶的水溶液。
优选的，所述恒温扩增酶包括重组酶聚合酶和切刻内切酶；进一步优选的，所述恒温扩增正向引物的碱性序列包含切刻内切酶的切刻位点CACTGC和保护碱基，正向引物序列为：SEQ ID NO.2；反向引物序列为：SEQ ID NO.3。
优选的，所述检测试纸包括胶体金试纸条。
本发明的第三个方面，提供了本发明所述的试剂盒在制备检测新型冠状病毒和/或检测新型冠状病毒引起的疾病的试剂中的应用。
优选的，新型冠状病毒引起的疾病包括新型冠状病毒肺炎。
本发明相对于现有技术具有如下优点：本发明开发了一套可居家自检的SARS-CoV-2检测方案。前期在SARS-CoV-2标准品DNA和RNA中进行了测试，具有很高的敏感性和特异性；对新冠患者鼻拭子和咽拭子抽取的核酸进行检测，5例可检测4例，且循环数为37的样本可检测，说明这种方法具有极高的敏感性。此外，我们开发了独特的基于磁珠吸附的核酸提取方法，实现了从核酸提取、核酸检测、结果读取的全套流程。这套方案不依赖于大型仪器，只需37℃和95℃两种温度，具备居家检测的条件。
附图说明
图1为本发明实施例1中基于磁珠富集和裂解液裂解的漱口水中细胞的核酸验证结果；
图2为本发明实施例2中探针及单链外切酶对探针的切割的结果；
图3为本发明实施例2中用新型冠状病毒DNA和RNA标准品检测结果；
图4为本发明实施例3中用新冠病人鼻拭子或咽拭子中纯化的核酸检测结果。
图5为本发明实施例4中用新冠病人漱口液进行检测结果。
具体实施方式
实施例1 用磁珠和裂解液释放细胞中的核酸并进行PCR验证
1.1、用清水漱口；
1.2、含入5ml RPMI1640培养基，漱口10s，将含入的培养基吐至50ml收集管中；
1.3、用吸管吸取1ml漱口水，加入2ml含有磁珠的B管中，颠倒混匀，室温静置10min；
1.4、将B管置于磁力架上，吸附10min，弃液体；
1.5从磁力架上取下B管，加入100μl裂解液，室温静置5min，95℃ 1min，取出后冷却至室温，在磁力架上吸附。
1.6、PCR验证人体细胞核酸：其中阴性对照的PCR模板为RPMI1640培养基，阳性对照1的PCR模板为HEK293基因组DNA，阳性对照组2的PCR模板为采用商业化试剂盒对口腔上皮细胞进行裂解得到的细胞核酸；实验组的PCR模板为磁珠和裂解液释放细胞核酸。
PCR体系：
Figure PCTCN2021109788-appb-000003
Figure PCTCN2021109788-appb-000004
其中，β-actin-F primer的碱基序列为：SEQ ID NO.4；β-actin-R primer的碱基序列为SEQ ID NO.5。
SEQ ID NO.4 GGACTTCGAGCAAGAGATGG
SEQ ID NO.5：AGCACTGTGTTGGCGTACAG
1.7、取10μl扩增产物在2％琼脂糖凝胶(含核酸染料)中电泳，凝胶拍照。
结果：实验组、阴性对照组、阳性对照组1、阳性对照组2的电泳结果见附图1。上述结果证明采用本发明提供的磁珠和裂解液法可以释放细胞内的核酸，其释放核酸的效果与商业化试剂盒效果几乎没有差别。
实施例2 检测新型冠状病毒(SARS-CoV-2)探针降解试验
2.1、制备探针，设计并修饰探针，探针核酸序列为：SEQ ID NO.1，其中5’端标记有地高辛修饰，3’端标记有生物素修饰。
2.2、阴性对照组(样本1)为水，实验组分别为：1pmol带有修饰的探针(样本2)，10pmol带有修饰的探针(样本3)，10pmol带有修饰的探针被单链外切酶Exo VII酶切后(样本4)。
2.3、取50μl 2.2中的产物，将胶体金试纸条插入产物中静置层析，5分钟内读取结果。
2.4、结果如附图2所示，在质控线(C)位置均有条带显示，说明实验成功；在检测线(T)位置，样本1没有条带为阴性，样本2有条带为阳性，样本3有条带为阳性，样本4没有条带为阴性。说明探针可直接检出，探针经外切酶切割后不可检出。
实施例3 检测新型冠状病毒(SARS-CoV-2)DNA标准品和RNA标准品3.1、制备检测样本：其中阴性对照组(样本1)采用水，实验组分别采用1000拷贝的SARS-CoV-2 DNA片段(样本2)、10000拷贝的SARS-CoV-2 RNA标准品(样本3)和1000拷贝的SARS-CoV-2 RNA标准品(样本4)。
3.2、第一次恒温扩增：以步骤1得到的检测样本为模板，使用RNA恒温快速扩增试剂盒(安普未来，WLRN8206KIT)进行恒温扩增，恒温扩增步骤参考试剂盒说明书。
反应体系如下：
Figure PCTCN2021109788-appb-000005
反应条件：37℃，12min。
其中，正向应物F为：SEQ ID NO.2；
反向引物R为：SEQ ID NO.3。
3.3、二次扩增：加入1μl Nb.BtsI、1μl Bst3.0、4μl dNTPs、37℃反应15min，95℃ 5min。
3.4、探针杂交：加入10μM标记的探针(实施例2提供的检测探针)2μl，95℃ 1min进行杂交，后缓慢降至室温(约15min)。
3.5、多余探针降解：取20μl杂交后产物，加入0.5μl Exo VII，37℃ 5min。
3.6、胶体金试纸条检测：加入60μl ddH2O，混匀后把胶体金试纸条插入到液体中，5分钟内读取结果。
3.7、结果如附图3所示，在质控线(C)位置均有条带显示，说明实验成功；在检测线(T)位置，样本1没有条带为阴性，样本2有条带为阳性，样本3有条带为阳性，样本4有条带为阳性。说明采用本发明提供的方法可以检测出SARS-CoV-2 DNA和SARS-CoV-2 RNA，且检测准确率100％。
实施例4 通过临床咽拭子样本检测新型冠状病毒(SARS-CoV-2)
4.1、采集5例新型冠状病毒肺炎疑似患者的咽拭子样本，临床核酸诊断结果为：样本1，阳性；样本2，阴性；样本3，阴性；样本4，阳性；样本5，阴性。
4.2、采用实施例1提供的方法提取咽拭子样本中的核酸。
4.3、以水为阴性对照组、1000拷贝的SARS-CoV-2 DNA片段为阳性对照组、步骤4.1获得的5例患者核酸为实验组，采用实施例3的方法进行检测。
4.4、结果如附图4所示，在质控线(C)位置均有条带显示，说明实验成功；在检测线(T)位置，阴性对照品没有条带为阴性，阳性对照品有条带为阳性，样本1有条带为阳性，样本2没有条带为阴性，样本3没有条带为阴性，样本4有条带为阳性，样本5没有条带为阴性。本发明提供的方法的检测疑似患者的结果与临床检测结果完全吻合，说明本发明具有检测新型冠状病毒的推广应用的潜能。
实施例5 通过漱口液样本检测新型冠状病毒(SARS-CoV-2)
5.1、采集4例新型冠状病毒肺炎确诊患者的漱口液样本，临床核酸诊断结果为：样本1，阳性；样本2，阳性；样本3，阳性；样本4，阳性。
5.2、采用实施例1提供的方法提取漱口液样本中的核酸。
5.3、以水为阴性对照组、1000拷贝的SARS-CoV-2 DNA片段为阳性对照组、步骤5.1获得的4例患者核酸为实验组，采用实施例3的方法进行检测。
5.4、结果如附图5所示，在质控线(C)位置均有条带显示，说明实验成功；在检测线(T)位置，阴性对照品没有条带为阴性，阳性对照品有条带为阳性，样本1有条带为阳性，样本2有条带为阴性，样本3有条带为阳性，样本4有条带为阳性。本发明提供方法的检测疑似患者的结果与临床检测结果完全吻合，说明本发明具有检测新型冠状病毒的推广应用的潜能。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
Figure PCTCN2021109788-appb-000006
Figure PCTCN2021109788-appb-000007

Claims (25)

  1. 一种新型冠状病毒快速检测方法，其特征在于，包括以下步骤：
    S1：获取待检测样本，采用磁珠吸附法获取样本中的待检测组分，并通过裂解法将吸附的细胞或病原体裂解，释放出细胞或病原体内的核酸；
    S2：将步骤S1得到的核酸样本进行多次恒温扩增，且最后一次恒温扩增为产生核酸单链的恒温扩增；
    S3：将步骤S2多次恒温扩增的单链产物与修饰的探针进行杂交；
    S4：将步骤S3杂交后的产物中加入特异性外切酶，切除多余的探针；
    S5：检测S4的产物。
  2. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S2还包括：向S1得到的RNA核酸样本中加入反转录酶，使RNA核酸样本反转录为DNA核酸样本。
  3. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S1中所述待检测样本包括漱口水、唾液、鼻拭子、咽拭子、痰液、刮片、尿液、粪便、机体分泌物、组织样本。
  4. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S1中所述磁珠吸附法使用的磁珠包括伴刀豆球蛋白A包被磁珠。
  5. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S3中所述探针为修饰的寡核苷酸探针，修饰为生物素修饰、地高辛修饰、异硫氰酸荧光素修饰、德克萨斯红修饰、磷酸化修饰、氨基修饰、巯基修饰、硫代修饰、荧光基团修饰、淬灭基团修饰中的一种或多种。
  6. 根据权利要求5所述的新型冠状病毒快速检测方法，其特征在于，所述探针的碱基序列为SEQ ID NO:1。
  7. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤 S4中所述特异性外切酶为核酸外切酶VII、λ核酸外切酶、T5核酸外切酶、T7核酸外切酶、核酸外切酶V、核酸外切酶III、RecJ1核酸外切酶、核酸外切酶I中的一种或多种。
  8. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S1所述的裂解法包括采用裂解液进行细胞裂解。
  9. 根据权利要求8所述的新型冠状病毒快速检测方法，其特征在于，所述裂解液包括10mM HEPES,0.3％SDS,0.5％IGEPAL-CA630,1.5mM MgCl2,0.1％焦碳酸二乙酯，10mM KCl，50mM Tris,10mM EDTA,1％RNA酶抑制剂，和1％蛋白酶的水溶液。
  10. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S2中所述的恒温扩增包括重组酶聚合酶扩增技术、重组酶辅助的扩增技术、环介导的等温扩增技术、解旋酶依赖的等温扩增技术、链置换的等温扩增技术、基于核酸序列的等温扩增技术、滚环扩增、切刻内切酶扩增反应。
  11. 根据权利要求10所述的新型冠状病毒快速检测方法，其特征在于，步骤S2所述的多次恒温扩增包括依次进行一次重组酶聚合酶扩增和一次切刻内切酶扩增。
  12. 根据权利要求11所述的新型冠状病毒快速检测方法，其特征在于，所述切刻内切酶扩增采用的切刻内切酶为Nb.BtsI、Nt.BspQI、Nt.CviPII、Nt.BstNBI、Nb.BsrDI、Nb.BtsI、Nt.AlwI、Nb.BbvCI、Nt.BbvCI、Nb.BsmI、Nb.BssSI、Nt.BsmAI中的一种或多种，所述的切刻内切酶扩增采用的链置换核酸聚合酶为Bst3.0 DNA聚合酶、Bst2.0 DNA聚合酶、Klenow DNA聚合酶、phi29 DNA聚合酶中的一种或多种。
  13. 根据权利要求12述的新型冠状病毒快速检测方法，其特征在于，多次 恒温扩增，正向引物的碱性序列包含切刻内切酶的切刻位点CACTGC和保护碱基。
  14. 根据权利要求1所述的新型冠状病毒快速检测方法，其特征在于，步骤S5中检测杂交产物的方法包括胶体金法、胶体碳法。
  15. 根据权利要求14所述的新型冠状病毒快速检测方法，其特征在于，检测杂交产物的方法为胶体金试纸条。
  16. 一种新型冠状病毒快速检测试剂盒，其特征在于，包括磁珠、细胞裂解液、恒温扩增引物、恒温扩增酶、探针、特异性外切酶、检测试纸。
  17. 根据权利要求16所述的试剂盒，其特征在于，所述磁珠包括伴刀豆球蛋白A包被磁珠。
  18. 根据权利要求16所述的试剂盒，其特征在于，所述探针为修饰的寡核苷酸探针，修饰为生物素修饰、地高辛修饰、异硫氰酸荧光素修饰、德克萨斯红修饰、磷酸化修饰、氨基修饰、巯基修饰、硫代修饰、荧光基团修饰、淬灭基团修饰中的一种或多种。
  19. 根据权利要求18所述的试剂盒，其特征在于，所述探针的碱基序列为SEQ ID NO:1。
  20. 根据权利要求16所述的试剂盒，其特征在于，所述特异性外切酶为核酸外切酶VII、λ核酸外切酶、T5核酸外切酶、T7核酸外切酶、核酸外切酶V、核酸外切酶III、RecJ1核酸外切酶、核酸外切酶I中的一种或多种。
  21. 根据权利要求16所述的试剂盒，其特征在于，所述细胞裂解液包括10mM HEPES,0.3％SDS,0.5％IGEPAL-CA630,1.5mM MgCl2,0.1％焦碳酸二乙酯，10mM  KCl，50mM Tris,10mM EDTA,1％RNA酶抑制剂，和1％蛋白酶的水溶液。
  22. 根据权利要求16所述的试剂盒，其特征在于，所述恒温扩增酶包括重组酶聚合酶和切刻内切酶。
  23. 根据权利要求22所述的试剂盒，其特征在于，所述恒温扩增正向引物的碱性序列包含切刻内切酶的切刻位点CACTGC和保护碱基。
  24. 根据权利要求16所述的试剂盒，其特征在于，所述检测试纸包括胶体金试纸条。
  25. 权利要求16-24任意一项所述的试剂盒在制备检测新型冠状病毒和/或检测新型冠状病毒引起的疾病的试剂中的应用。
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