CN111378787A - Novel coronavirus detection method - Google Patents
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- CN111378787A CN111378787A CN202010267246.0A CN202010267246A CN111378787A CN 111378787 A CN111378787 A CN 111378787A CN 202010267246 A CN202010267246 A CN 202010267246A CN 111378787 A CN111378787 A CN 111378787A
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 3
- 238000011529 RT qPCR Methods 0.000 claims abstract 3
- 238000000034 method Methods 0.000 claims description 18
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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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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
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Abstract
The invention discloses a novel coronavirus detection method, which comprises the following steps: preparing qPCR reaction liquid, enzyme mixed liquid, amplification primer and probe mixed liquid, constructing a reaction system, amplifying, analyzing a fluorescent signal and judging whether a target gene exists in a specimen. The detection method combines two genes with reverse transcription and qPCR operation, can reduce the omission caused by the extremely low virus content in the specimen in the initial infection stage and the false detection caused by other coronaviruses, and improves the speed and the accuracy of the detection.
Description
Technical Field
The invention relates to a virus detection method, in particular to a detection method for novel coronavirus.
Background
Coronaviruses are RNA viruses with envelope and linear single-strand positive strand genomes, and are viruses susceptible to a large group of humans and various animals widely existing in nature. The novel coronavirus (SARS-CoV-2) is the 7 th coronavirus which is known to infect human, and the disease condition is named as COVID-19. After human beings are infected by coronavirus, respiratory diseases are usually more obvious, the diseases are more acute, the symptoms are more complex, multiple organ failures of the whole body can be caused, and the human bodies can be damaged to different degrees or even endanger the life.
The accurate detection aiming at SARS-CoV-2 is crucial to clinical early discovery, early diagnosis, early isolation and early treatment. The new crown pneumonia epidemic situation can be effectively prevented and controlled by effectively judging the infected people and preventing the virus from being spread again.
The diagnosis of COVID-19 cannot be confirmed by patient symptoms, CT examination or antibody kits, and only by real-time fluorescent quantitative PCR (qPCR) detection of viral nucleic acid, higher accuracy is achieved. The main principle of qPCR is to design specific oligonucleotide probes for a certain section of specific gene sequence of the virus, and to add fluorescent luminescent groups and quenching groups on two sides of the oligonucleotide probes respectively, so that a sample to be detected is in a non-fluorescent state. During amplification, DNA polymerase uses exonuclease activity to degrade fluorescent probes, thereby generating fluorescence at a specific wavelength. If the sample to be detected is infected with virus, the specific DNA sequence is exponentially increased along with PCR amplification, the more target genes are amplified in the sample to be detected, the stronger the accumulated fluorescent signal is, and in the sample without virus infection, the fluorescent signal cannot be enhanced due to the fact that the target genes are not amplified.
In the detection process, false positive and false negative phenomena occur when the detection is influenced by other coronavirus or the virus amount in a patient sample is very small, so that false detection and missed detection are caused. Only by solving the above problems, a reliable qPCR nucleic acid detection result can be obtained quickly and efficiently.
Disclosure of Invention
The invention aims to provide a novel coronavirus detection method, which can reduce false negative and false positive results caused by missed detection and false detection and improve the accuracy and speed of detection.
The detection method of the novel coronavirus comprises the following steps:
1) preparing qPCR reaction liquid, enzyme mixed liquid and primer probe mixed liquid;
2) constructing a reaction system: 9 mu L of reaction solution, 6 mu L of enzyme mixed solution, 5 mu L of primer probe mixed solution and 5 mu L of sample to be detected;
3) the following amplification steps were input and fluorescence signals were detected using FAM, VIC, Cy5 channels: circulating for 45 times at 50 deg.C for 15min, 95 deg.C for 3min, 95 deg.C for 15s, and 58 deg.C for 30 s.
The primer probe mixed solution comprises a primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 for amplifying ORF1ab gene, a probe shown in SEQ ID NO.3, a primer pair shown in SEQ ID NO.4 and SEQ ID NO.5 for amplifying N gene and a probe shown in SEQ ID NO.6, wherein the concentration of the primers and the concentration of the probes are both 2.5 mu M.
Furthermore, the primer probe mixed solution also comprises a primer pair shown in SEQ ID NO.7 and SEQ ID NO.8 of the internal reference GADPH for amplification and a probe shown in SEQ ID NO.9, and the concentration of the primer and the concentration of the probe are both 2.5 mu M.
The enzyme mixture comprises: 0.04U/. mu.L UDG enzyme, 0.075U/. mu.L Taq enzyme, 0.05U/. mu.L reverse transcriptase.
The reaction solution comprises: pH8.5, 20mM Tris-HCl, 40mM KCl, 0.3% TritonX-100, 4mM MgCl22% DMSO, 0.4mM dATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dUTP, 0.2mM dTTP, 2M betaine.
The 5 'end of the probe shown in SEQ ID NO.3 is modified with a fluorescent group FAM, and the 3' end is modified with a quenching group BHQ-1.
The probe shown in SEQ ID NO.6 is modified with a fluorescent group VIC at the 5 'end and a quenching group BHQ-1 at the 3' end.
The probe shown in SEQ ID NO.9 is modified with a fluorescent group Cy5 at the 5 'end and a quenching group BHQ-3 at the 3' end.
Compared with other methods, the detection method has the following beneficial effects: 1) reverse transcription and qPCR are carried out in the same system, so that the experimental efficiency is improved, and pollution caused by repeatedly opening a tube cover is avoided; 2) ORF1ab gene and N gene can be detected simultaneously in the same reaction tube, thereby reducing false negative and false positive rate and improving accuracy and sensitivity; 3) three-channel detection can be used for detecting a target gene and judging whether the nucleic acid extraction and amplification processes are effective or not and whether the quality of a sample is problematic or not.
Detailed Description
Example 1 patient sample testing.
1) And preparing a solution required by the reaction.
The qPCR reaction solution was prepared according to the components and concentrations shown in Table 1.
An enzyme mixture was prepared according to the components and concentrations shown in Table 2.
Preparing a primer and probe mixed solution, wherein the concentration of the primer and the probe is 2.5 mu M, and the primer and the probe mixed solution comprise the following components:
amplifying primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 and probes shown in SEQ ID NO.3 of ORF1ab gene;
amplifying primer pairs shown in SEQ ID NO.4 and SEQ ID NO.5 and probes shown in SEQ ID NO.6 of the N gene;
amplifying primer pairs shown as SEQ ID NO.7 and SEQ ID NO.8 of the internal reference GADPH and a probe shown as SEQ ID NO. 9;
reverse transcription primer Oligo dT, random primer.
2) The method comprises the steps of carrying out nucleic acid detection on retained specimens of patients diagnosed in new crown department of the department of pulmonaries of Shanxi medical university, and respectively selecting 10 positive specimens and 3 negative specimens. The reaction system is shown in Table 3.
3) The amplification procedure shown in Table 4 was followed and the fluorescent signal was detected.
The 5 'end of the probe shown in SEQ ID NO.3 is modified with a fluorescent group FAM, and the 3' end is modified with a quenching group BHQ-1; the 5 'end of the probe shown in SEQ ID NO.6 is modified with a fluorescent group VIC, and the 3' end is modified with a quenching group BHQ-1; the probe shown in SEQ ID NO.9 is modified with a fluorescent group Cy5 at the 5 'end and a quenching group BHQ-3 at the 3' end. The results of three-channel fluorescence detection are shown in the table 5, and the coincidence rate of the results of 10 positive samples and 3 negative samples reaches 100 percent.
Example 2 the detection method of the present invention is a standard of judgment.
And if the Ct values of the positive control substances, namely FAM and VIC channels, are less than or equal to 31 and have S-type amplification curves, and the negative control substances are not amplified, the parallel experiment is effective. The judgment criteria are shown in Table 6.
According to the detection results of the 3 channels, the following results can be judged:
ORF1ab gene and N gene are both positive, and the result is determined to be positive for SARS-CoV-2.
If ORF1ab gene is positive only, repeated measurement is required, and if the result is positive, SARS-CoV-2 is determined to be positive.
If only the N gene is positive and still positive after repeated determination, the coronavirus may be other closely derived coronavirus.
ORF1ab gene and N gene were negative, and they were judged to be SARS-CoV-2 negative.
Example 3 test methods in-batch precision calculations.
The method provided by the invention is used for detecting the N gene standard substance of the national measurement institute. The batch precision is calculated, and table 7 shows that the batch precision of the method is less than 2%, which indicates that the method has good detection stability and can be used for detecting a large number of patients.
Sequence listing
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Claims (9)
1. A method for detecting novel coronavirus is characterized in that primer probe mixed liquor is prepared, wherein the primer probe mixed liquor comprises the following steps: the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 of amplified ORF1ab gene, the probe shown in SEQ ID NO.3, the primer pair shown in SEQ ID NO.4 and SEQ ID NO.5 of amplified N gene and the probe shown in SEQ ID NO. 6.
2. The method for detecting a novel coronavirus according to claim 1, further comprising the steps of:
1) preparing qPCR reaction liquid and enzyme mixed liquid;
2) constructing a reaction system: 9 mu L of reaction solution, 6 mu L of enzyme mixed solution, 5 mu L of primer probe mixed solution and 5 mu L of sample to be detected;
3) the following amplification steps were input and the fluorescence signal was detected: circulating for 45 times at 50 deg.C for 15min, 95 deg.C for 3min, 95 deg.C for 15s, and 58 deg.C for 30 s.
3. The method for detecting a novel coronavirus according to claim 1, wherein the primer-probe mixture further comprises a primer set represented by SEQ ID No.7 and SEQ ID No.8 and a probe represented by SEQ ID No.9 for amplifying the internal control GADPH.
4. The method for detecting a novel coronavirus according to claim 1, wherein the enzyme mixture solution comprises: 0.04U/. mu.L UDG enzyme, 0.075U/. mu.L Taq enzyme, 0.05U/. mu.L reverse transcriptase.
5. The method of detecting a novel coronavirus according to claim 1, wherein the reaction solution comprises: pH8.5, 20mM Tris-HCl, 40mM KCl, 0.3% TritonX-100, 4mM MgCl22% DMSO, 0.4mM dATP, 0.4mM dCTP, 0.4mM dGTP, 0.4mM dUTP, 0.2mM dTTP, 2M betaine.
6. The method for detecting a novel coronavirus according to claim 1, wherein the probe represented by SEQ ID NO.3 is modified at the 5 'end with a fluorescent group FAM and at the 3' end with a quencher group BHQ-1.
7. The method for detecting a novel coronavirus according to claim 1, wherein the probe represented by SEQ ID NO.6 is modified at the 5 'end with a fluorescent group VIC and at the 3' end with a quencher group BHQ-1.
8. The method for detecting a novel coronavirus according to claim 3, wherein the probe represented by SEQ ID NO.9 is modified at the 5 '-end with a fluorescent group Cy5 and at the 3' -end with a quencher group BHQ-3.
9. The method for detecting a novel coronavirus according to claim 1 or 3, wherein the concentration of each of the primer and the probe is 2.5. mu.M.
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| CN202010267246.0A CN111378787A (en) | 2020-04-08 | 2020-04-08 | Novel coronavirus detection method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029910A (en) * | 2020-09-30 | 2020-12-04 | 东南大学 | Nucleic acid detection method for SARS-CoV-2 virus |
| CN114317822A (en) * | 2021-12-28 | 2022-04-12 | 山西大学 | Multiplex fluorescence quantitative PCR method for detecting new coronavirus nucleic acid |
-
2020
- 2020-04-08 CN CN202010267246.0A patent/CN111378787A/en active Pending
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| Title |
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| VICTOR M CORMAN ET AL.: "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR", 《EURO SURVEILL》 * |
| YANG ZHANG ET AL.: "Genetic and immune features of resectable malignant brainstem gliomas", 《ONCOTARGET》 * |
| 丁晓燕: "2种国产新型冠状病毒核酸检测试剂检测结果的比较与分析", 《分子诊断与治疗杂志》 * |
| 国家卫生健康委员会: "新型冠状病毒的实验室检测", 《新型冠状病毒肺炎实验室检测技术指南（第四版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029910A (en) * | 2020-09-30 | 2020-12-04 | 东南大学 | Nucleic acid detection method for SARS-CoV-2 virus |
| CN112029910B (en) * | 2020-09-30 | 2022-06-17 | 东南大学 | Nucleic acid detection method for SARS-CoV-2 virus |
| CN114317822A (en) * | 2021-12-28 | 2022-04-12 | 山西大学 | Multiplex fluorescence quantitative PCR method for detecting new coronavirus nucleic acid |
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Application publication date: 20200707 |