CN111206122A - A novel coronavirus nucleic acid detection kit - Google Patents
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- CN111206122A CN111206122A CN202010262236.8A CN202010262236A CN111206122A CN 111206122 A CN111206122 A CN 111206122A CN 202010262236 A CN202010262236 A CN 202010262236A CN 111206122 A CN111206122 A CN 111206122A
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- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 24
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 24
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 24
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 7
- 239000012498 ultrapure water Substances 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000013641 positive control Substances 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 2
- 229960003237 betaine Drugs 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 102100034343 Integrase Human genes 0.000 claims 1
- 238000011529 RT qPCR Methods 0.000 claims 1
- 208000025721 COVID-19 Diseases 0.000 abstract description 4
- 239000007850 fluorescent dye Substances 0.000 abstract description 3
- 239000013558 reference substance Substances 0.000 abstract description 2
- 108700026220 vif Genes Proteins 0.000 abstract description 2
- 241000700605 Viruses Species 0.000 description 14
- 241000282414 Homo sapiens Species 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 101150013191 E gene Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 108010076039 Polyproteins Proteins 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 239000013610 patient sample Substances 0.000 description 1
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- 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
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- 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
- C12Q1/6851—Quantitative amplification
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Abstract
本发明公开了一种新型冠状病毒核酸检测试剂盒，包括用于扩增ORF1ab基因的核酸扩增试剂、对照品和超纯水。所述核酸扩增试剂包括SEQ NO.1~2所示的引物和SEQ NO.3所示的探针混合液，所述探针5’端修饰荧光基团FAM，3’端修饰淬灭基团BHQ‑1。本发明所述试剂盒通过特异性的引物及荧光探针，检测标本中是否存在ORF1ab基因，以此判断被检人是否患有COVID‑19。The invention discloses a novel coronavirus nucleic acid detection kit, which includes a nucleic acid amplification reagent for amplifying the ORF1ab gene, a reference substance and ultrapure water. The nucleic acid amplification reagent includes the primers shown in SEQ NO.1 to 2 and the probe mixture shown in SEQ NO.3, the 5' end of the probe is modified with a fluorescent group FAM, and the 3' end is modified with a quencher group Mission BHQ‑1. The kit of the present invention detects whether the ORF1ab gene exists in the specimen through specific primers and fluorescent probes, thereby judging whether the tested person has COVID-19.
Description
Technical Field
The invention relates to a virus detection kit, in particular to a novel coronavirus nucleic acid detection kit.
Background
Coronavirus is a large group of viruses widely existing in nature, is a linear single-stranded positive-strand RNA virus, is an important pathogen of diseases of many domestic animals and pets including human beings, and can cause various respiratory and intestinal diseases, and coronavirus such as MERS and SARS can cause serious respiratory diseases. The novel coronavirus (2019-nCoV) which is outbreaked in the early 2020 belongs to the genus coronavirus in the family coronavirus, and is the 7 th coronavirus which is known to infect human at present. The disorder is designated COVID-19.
The virus has a unique gene sequence, and the presence of the virus in a patient can be judged by detecting the virus nucleic acid in the patient. However, viruses in human bodies may disappear along with the rehabilitation of patients, so nucleic acid detection is an important index for clinically judging whether patients are infected or cured. Therefore, the rapid detection of the novel coronavirus is an important means for effectively judging infected people and preventing the virus from being spread again, is very important for clinical early discovery, early diagnosis, early isolation and early treatment, and can effectively prevent and control the new coronary pneumonia epidemic situation.
At present, the virus nucleic acid detection method usually adopts a fluorescent quantitative PCR method (qPCR). qPCR takes a unique gene sequence of a virus as a detection target, a specific oligonucleotide probe is designed aiming at the specific gene sequence, and a fluorescent luminescent group and a quenching group are respectively added on two sides of the specific oligonucleotide probe. In amplification, the target gene is exponentially increased, and the DNA polymerase degrades the fluorescent probe using exonuclease activity, thereby generating a fluorescent signal of a specific wavelength. Most of the current virus detection kits select the sequences of N gene, E gene and ORF1ab gene of virus as target genes, wherein the E gene encodes virus envelope protein E, the N gene encodes virus nucleocapsid protein N, and the ORF1ab gene encodes polyprotein pp1 ab. If the target gene is present in the sample, the more fragments that are amplified, the stronger the fluorescent signal obtained by accumulation. In the sample without the virus, the fluorescence signal was not increased because the target gene was not amplified.
However, the accuracy of nucleic acid detection depends on various factors, and the detection results may vary greatly from sample to sample in the same patient. In addition, as the course of the disease changes, the amount of virus in the patient also changes dynamically. Therefore, the development of efficient and sensitive kits is a key factor for rapid detection.
Disclosure of Invention
The purpose of the present invention is to provide a novel coronavirus nucleic acid detection kit for determining whether a test subject has COVID-19 by detecting the presence or absence of ORF1ab gene in a sample using specific primers and fluorescent probes.
The novel coronavirus nucleic acid detection kit comprises a nucleic acid amplification reagent for amplifying novel coronavirus ORF1ab gene, a reference substance and ultrapure water.
The nucleic acid amplification reagent comprises a primer shown in SEQ NO. 1-2 and a probe mixed solution shown in SEQ NO. 3.
The reaction solution comprises the following components: pH8.5, 20mM Tris-HCl, 40mM KCl, 0.3% TritonX-100, 4mM MgCl210% DMSO, 0.4mM dATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dUTP, 0.2mM dTTP, 2M betaine, 0.04U/. mu.L UDG enzyme, 0.075U/. mu.L Taq enzyme, 0.05U/. mu.L reverse transcriptase, 1. mu.M Random primer, 1. mu.M.
The concentrations of the primers shown in SEQ ID NO.1 and SEQ ID NO.2 and the probe shown in SEQ ID NO.3 are both 2.5 mu M.
The 5 'end of the probe is modified with a fluorescent group FAM, and the 3' end of the probe is modified with a quenching group BHQ-1.
The control product comprises a negative control product and a positive control product.
The use method of the novel coronavirus nucleic acid detection kit comprises the following steps:
1) prepare 10 μ L reaction: 5 mul of reaction liquid, 2 mul of primer-probe mixed liquid, 2 mul of template RNA and 1 mul of ultrapure water;
2) after mixing uniformly, putting the mixture into a qPCR instrument, and setting the fluorescence type;
3) amplification was started after setting the following cycle: the amplification is finished after 40 cycles of 50 ℃ for 10min, 95 ℃ for 3min,95 ℃ for 15s and 58 ℃ for 30 s.
The kit has the following beneficial effects: 1) a dUTP/UDG anti-pollution system is introduced, so that false positive results caused by cross reaction can be effectively avoided; 2) the specific primer probe designed aiming at the novel coronavirus ORF1ab gene and the special buffer system can ensure that the DNA polymerase can exert the maximum effect and improve the reaction efficiency. 3) The method can obtain a wider linear range, has more accurate target gene quantification, good repeatability and high reliability, and is suitable for detecting trace RNA such as RNA virus.
Detailed Description
Example 1 methods of using the novel coronavirus nucleic acid detection kit.
(1) And taking the reaction solution, the primer probe solution and the ultrapure water out of a refrigerator at the temperature of-20 ℃, thawing at room temperature and mixing uniformly.
(2) The reaction system shown in table 1 was placed in a qPCR reaction tube, shaken, mixed and centrifuged briefly.
(3) And setting a parallel test of a negative control and a positive control, wherein the negative control replaces the template RNA with ultrapure water, and the positive control replaces the template RNA with a positive control product in the reagent kit.
(4) Parameters such as the volume of the reaction system, the type of fluorescence and the like are set on a qPCR instrument, and reaction conditions shown in Table 2 are input.
(5) And (4) interpretation of results: and judging whether the sample contains the novel coronavirus sequence according to the output copy number per milliliter.
The output result is negative when no Ct value exists or the Ct value is more than 40; a positive may be reported when the output Ct value is < 37; the experiment can be repeated when the output Ct value is between 37 and 40, if the Ct value is less than 40, the amplification curve has obvious peak, the sample is judged to be positive, otherwise, the sample is negative.
Example 2 nucleic acid detection in patient samples.
The method of example 1 is used to perform nucleic acid detection on the retained specimen of the patient diagnosed in Xinguan diagnosis of Feicuo Hospital, Shanxi medical university, 10 COVID-19 patient specimens and 3 healthy person specimens respectively, the results are shown in Table 3, the Ct values of the patient specimens 1-10 are all less than 37, all specimens are judged to be positive, the healthy person specimens 11-13 have no Ct, and the detection rate reaches 100%.
Sequence listing
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Claims (6)
1. A novel coronavirus nucleic acid detection kit comprises a nucleic acid amplification reagent for amplifying novel coronavirus ORF1ab genes, and is characterized in that the nucleic acid amplification reagent comprises a mixed solution of primers shown in SEQ ID NO. 1-2 and a probe shown in SEQ ID NO. 3.
2. The detection kit according to claim 1, wherein the probe is modified with a fluorescent group FAM at the 5 'end and a quencher group BHQ-1 at the 3' end.
3. The detection kit according to claim 1, wherein the nucleic acid amplification reagent further comprises a reaction solution consisting of: pH8.5, 20mM Tris-HCl, 40mM KCl, 0.3% TritonX-100, 4mM MgCl210% DMSO, 0.4mM dATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dUTP, 0.4mM dTTP, 2M betaine, 0.04U/. mu.L UDG enzyme, 0.075U/. mu.L Taq enzyme, 0.05U/. mu.L reverse transcriptase, 1. mu.M Random primer, 1. mu.M Oligo dT.
4. The detection kit according to claim 1, wherein the concentrations of the primers shown by SEQ ID NO.1 and SEQ ID NO.2 and the concentration of the probe shown by SEQ ID NO.3 are 2.5. mu.M.
5. The test kit according to claim 1, further comprising a control and ultrapure water, wherein the control comprises a negative control and a positive control.
6. The method for using the novel coronavirus nucleic acid detection kit of claim 1, comprising the steps of:
1) prepare 10 μ L reaction: 5 mul of reaction liquid, 2 mul of primer-probe mixed liquid, 2 mul of template RNA and 1 mul of ultrapure water;
2) after mixing uniformly, putting the mixture into a qPCR instrument, and setting the fluorescence type;
3) amplification was started after setting the following cycle: the amplification is finished after 40 cycles of 50 ℃ for 10min, 95 ℃ for 3min,95 ℃ for 15s and 58 ℃ for 30 s.
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| CN202010262236.8A CN111206122A (en) | 2020-04-06 | 2020-04-06 | A novel coronavirus nucleic acid detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690772A (en) * | 2020-06-15 | 2020-09-22 | 桂林优利特医疗电子有限公司 | New coronavirus nucleic acid detection kit, preparation method and application |
| CN112553375A (en) * | 2020-12-22 | 2021-03-26 | 武汉艾迪康医学检验所有限公司 | Respiratory virus detection kit and method |
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| US7220852B1 (en) * | 2003-04-25 | 2007-05-22 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Coronavirus isolated from humans |
| CN107523642A (en) * | 2017-10-20 | 2017-12-29 | 苏州旷远生物分子技术有限公司 | A kind of chain reaction of multiple reverse transcription polymerase detection reagent buffer solution and its application |
| CN109371174A (en) * | 2018-12-20 | 2019-02-22 | 江苏和创生物科技有限公司 | Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit |
-
2020
- 2020-04-06 CN CN202010262236.8A patent/CN111206122A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7220852B1 (en) * | 2003-04-25 | 2007-05-22 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Coronavirus isolated from humans |
| CN107523642A (en) * | 2017-10-20 | 2017-12-29 | 苏州旷远生物分子技术有限公司 | A kind of chain reaction of multiple reverse transcription polymerase detection reagent buffer solution and its application |
| CN109371174A (en) * | 2018-12-20 | 2019-02-22 | 江苏和创生物科技有限公司 | Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| 匡慧慧等: "新型冠状病毒实验室核酸检测方法及实践", 《中华医院感染学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690772A (en) * | 2020-06-15 | 2020-09-22 | 桂林优利特医疗电子有限公司 | New coronavirus nucleic acid detection kit, preparation method and application |
| CN112553375A (en) * | 2020-12-22 | 2021-03-26 | 武汉艾迪康医学检验所有限公司 | Respiratory virus detection kit and method |
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Application publication date: 20200529 |