CN111334614A - Method for detecting novel coronavirus by RT-qPCR technology - Google Patents
Method for detecting novel coronavirus by RT-qPCR technology Download PDFInfo
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Abstract
The invention provides a method for detecting novel coronavirus by adopting RT-qPCR technology, which comprises the following steps: 1) designing and synthesizing primers and probe primers; 2) preparing a reaction system comprising a reaction solution and a probe primer; 3) performing RT-qPCR reaction by using the nucleic acid of a sample to be detected and the recombinant plasmid as templates; 4) and collecting fluorescence signals to perform result analysis and reaction system sensitivity test. When the Ct values detected by the FAM channel and the HEX channel are both less than 37, the sample contains the novel coronavirus (SARS-Cov-2) and is positive. The technical scheme provided by the invention shortens the setting time, can sensitively and stably amplify the RNA target, has strong specificity, high sensitivity, good repeatability and simple and quick operation, provides an effective method for quickly detecting the novel coronavirus, and is suitable for clinical and scientific research popularization and application.
Description
Technical Field
The invention belongs to the technical field of virus molecular diagnosis, and particularly relates to a method for detecting a novel coronavirus by using an RT-qPCR (reverse transcription-quantitative polymerase chain reaction) technology.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19), abbreviated as 'novel coronavirus pneumonia', is named as '2019 coronavirus Disease' by the world health organization, and refers to pneumonia caused by infection of 2019 novel coronavirus (SARS-Cov-2), is identified as a beta type coronavirus (betaaconavirus) containing single-stranded positive-strand RNA (ssRNA), has a structure extremely similar to severe acute respiratory syndrome SARS, has about 70% and 40% sequence similarity with SARSr-CoV and MERSR-CoV, and has the whole genome sequence consistency of 96% with bat Virus TG 13. The main sequences of nucleic acids include E (envelope protein gene), M (membrane protein gene), N (nuclear associated protein gene), S (spike protein gene), ORF (open reading frame) and RdRp (RNA-dependent RNA polymerase gene). The virus can spread infection among people by means of spray, aerosol, feces mouth and the like. The novel coronavirus (SARS-CoV-2) can infect human respiratory epithelial cells through the molecular mechanism of its surface S-protein interaction with the human ACE2 receptor.
At present, there are many clinical diagnosis methods aiming at coronavirus, such as chest CT scanning, molecular diagnosis technology, immunochromatography and the like. The CT scanning technology is convenient but has insufficient specificity, and can assist in diagnosis; the immunochromatography method is simple to operate, but has poor specificity and sensitivity, is easy to cause result false negative and false positive, and is not suitable for clinical diagnosis; compared with the molecular diagnosis technology, the molecular diagnosis technology has high sensitivity and strong specificity, and is suitable for clinical diagnosis and diagnosis of the novel coronavirus. RT-qPCR technology, combining with quantitative fluorescent technology qPCR (quantitative real-time PCR, qPCR) reverse transcription PCR technology. Non-specific dye or specific probe combined with double-stranded DNA can be added in the PCR process to monitor the target DNA amplification process in real time. One common parameter is the Ct value, also called threshold cycle, which is the number of cycles that the fluorescence signal starts to enter the inflection point of the exponential growth phase from the background during the PCR cycle, i.e. the number of cycles that the fluorescence signal in each reaction tube passes through when it reaches the set threshold. The Ct value of each template is linear with the logarithm of the starting copy number of the template, i.e., the more the starting copy number, the smaller the Ct value. The fluorescence labeling probe is an oligonucleotide, two ends of the fluorescence labeling probe are respectively labeled with a report fluorescent group and a quenching fluorescent group, when the probe is complete, a fluorescence signal emitted by the report group can be absorbed by the quenching group, so that no fluorescence signal is generated, the probe is combined with any one DNA single strand, when PCR begins to amplify, the Taq enzyme cuts and degrades the probe, so that the report group is separated from the quenching group, and the fluorescence signal is detected by a fluorescence detection system. 1 fluorescent molecule is formed for each amplified 1 DNA chain, so that the equal proportion increase of a fluorescent signal and a PCR product is realized, and the aim of quantitative detection is fulfilled. In a PCR reaction system, excessive SYBR fluorescent dye is added, and after the SYBR fluorescent dye is specifically doped into a DNA double strand, a fluorescent signal is emitted, while SYBR dye molecules which are not doped into the strand do not emit any fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous. The RT-qPCR technology has the advantages of strong specificity, high sensitivity, good repeatability, accurate quantification, high speed, full-closed reaction and the like, is widely applied to aspects of basic research, disease research, tumor diagnosis and the like, and becomes an important tool in molecular biology research.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides an RT-qPCR detection method for detecting novel coronavirus, and the detection method has high specificity and sensitivity.
Preferably, the invention designs 2 primers with high efficiency and strong specificity according to the highly conserved gene ORF1ab and N gene of the genome sequence of the novel coronavirus (SARS-Cov-2), and 2 different probes can be subjected to double-color fluorescence labeling (ORF 1ab gene and N gene), so that the accuracy and specificity of the detection result are improved.
Preferably, the invention selects high-efficiency reverse transcriptase and DNA polymerase, greatly increases the reverse transcription efficiency and the amplification efficiency, shortens the detection time to 45 minutes at most, and reduces the detection time so as to increase the detection efficiency.
Preferably, the reagent combination is optimized, and the reaction buffer is matched under the condition of ensuring the enzyme activity, so that the operation steps of detection personnel are reduced, and the detection result error caused by misoperation in detection is reduced.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that: comprises the following steps.
First, synthesis of primers, artificially synthesized RNA, and positive control plasmid: two pairs of primers and probes are designed aiming at the N gene of the conserved region of the novel coronavirus and the ORF1ab gene. In vitro synthesis of RNA segment and DNA plasmid of target gene of novel coronavirus.
Secondly, respectively taking RNA nucleic acid extracted from the sample to be tested, artificially synthesized RNA and positive control plasmid as templates, carrying out qPCR amplification reaction by using specific primers and probes, and simultaneously selecting a FAM channel and a HEX channel for carrying out an experiment by using an instrument channel:
(1) the 20 μ L reaction system for the qPCR amplification reaction was: RNA 5. mu.l (0.01 pg-1. mu.g), primer probe (10. mu.M) 2. mu.l, reaction solution 13. mu.l;
(2) the qPCR amplification parameters were: keeping at 50 deg.C for 5min and 94 deg.C for 2 min; denaturation at 94 ℃ for 5s, annealing at 55 ℃ for 10s, for 40 cycles;
(3) judging the identification result: if the Ct values detected by the FAM channel and the HEX channel are both less than 37, the nucleic acid is judged to be positive by the novel coronavirus (SARS-CoV-2); if the Ct value detected by the FAM channel and the HEX channel has no value or the Ct value =40, and the detection result of the positive control is positive, the result is judged to be negative by the novel coronavirus (SARS-CoV-2) nucleic acid; if the CT value of the sample to be detected is between 37 and 40, the sample is recommended to be detected again after the nucleic acid is extracted again. If the repeated detection result shows that the Ct value is less than 40 and the amplification curve has obvious peak, the nucleic acid is judged to be positive by the novel coronavirus (SARS-CoV-2); otherwise, the result is negative.
Step three, sensitivity test of a reaction system: when the concentration is gradually reduced from 1/10 to 1/10000 by performing fluorescence PCR detection on positive SARS-CoV-2 nucleic acid, the cycle number (Ct) of the RFU result reaching the threshold value is less than 35. Meanwhile, under the basic condition that the initial value of the RNA concentration of a positive patient is 9.5ng/ul, the concentration dilution ratio reaches 1000000 times, the result that the cycle number (Ct) is lower than 37 is presented, the lowest detection value of the novel coronavirus can reach 1/1000000, and the method has good sensitivity.
Further, the sequences of the specific primers and probes in the first step are shown in FIG. 5.
Further, the DNA sequence of the recombinant positive control plasmid synthesized in the first step is,
ORF1ab gene:
AGACAGTTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGTAGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAACTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCTAAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGTTCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTCCGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGTTGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCTCAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATGCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTATTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTG
n gene:
TTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGT
the beneficial results of the invention are: the minimum concentration of RNA sample used for detection of SARS-CoV-2 virus was adjusted to 1/1000000 (initial value set at 9-10 ng/ul). The Cycle threshold (Ct) values measured by the applicability of clinical positive samples for detecting SARS-CoV-2 are all lower than 37, and the sensitivity test result also shows that the method has good sensitivity. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology adopting the TaqMan probe is better than SYBRGreen in specificity, has a specific probe and strong result characteristics; by adopting one-step RT-qPCR, RNA reverse transcription and PCR reaction are completed in the same tube in a totally closed state, thereby avoiding environmental pollution and false positive caused by the environmental pollution. Therefore, the detection method is suitable for any scientific research laboratory colleges and universities, medical and health units and the like. In conclusion, the method provided by the invention has the advantages of strong specificity, high sensitivity, good repeatability, simple and convenient operation and high detection speed.
Drawings
FIG. 1 shows the result of fluorescence qPCR detection of RNA fragment (130 ng/ul) of novel artificially synthesized coronavirus (SARS-CoV-2) after fluorescent quantitation at 7 different dilution concentrations; it can be seen that after the gradient dilution, the curve moves backwards in equal proportion.
FIG. 2 shows the positive patient with a nucleic acid RNA quantitation concentration of 9.5 ng/. mu.L at the following dilution ratios:
A=1：1000，
B=1：10000，
C=1：100000，
D=1：1000000
the qPCR results showed that ct values were still less than 37 at gradient dilutions to 1/1000000.
FIG. 3 shows the qPCR results for two positive patient samples tested using the finished kit of the invention. The sample of each patient was subjected to 2 replicates, two experiments were performed simultaneously, and a positive control, a negative control, patient 1 and patient 2 were performed simultaneously, showing that each set of experiments had good reproducibility, the positive control was very strong, and the negative control had no readings.
FIG. 4 is a genome map of the whole genome of the novel coronavirus.
FIG. 5 shows the nucleotide sequences, amplified fragment lengths and the locations of the viral genomes of the primers and probes used in the present invention.
Detailed Description
The invention is further described with reference to the accompanying drawings and the detailed description.
An RT-qPCR method for rapidly detecting novel coronavirus comprises the following steps.
Instruments and reagents
Fluorescent light quantitative PCR instrument (Bio-Rad CFX96 touch),
a PCR tube which is free from nuclease and has a PCR tube cover with good light transmittance,
0-2. mu.L of pipette gun (Eppendorf),
1-10. mu.L of pipette gun (Eppendorf),
2-20. mu.L of pipette gun (Eppendorf),
10-100. mu.L pipette (Eppendorf),
20-200. mu.L pipette gun (Eppendorf),
200-,
viral RNA nucleic acid extraction kit (Qiagen),
the nucleic acid detection kit of the invention.
(II) preparing a sample and extracting nucleic acid: for clinical samples, collected nasal and pharyngeal swabs and other samples are extracted and purified by a virus RNA extraction kit to obtain SARS-CoV-2 virus nucleic acid RNA (the step at least needs to be carried out in a P3 laboratory to reduce pollution source spread), and the SARS-CoV-2 virus nucleic acid RNA is stored at the temperature of minus 40 ℃ to minus 80 ℃ for later use.
(III) synthesis of primers and standard plasmids: two pairs of primers and probes are designed aiming at the N gene of the conserved region of the novel coronavirus and the ORF1ab gene. In vitro synthesizing new crown DNA sequence to synthesize standard recombinant plasmid. Specific primer and probe information is shown in FIG. 5.
And (IV) carrying out RT-qPCR reaction by taking the extracted sample RNA and the positive plasmid as templates.
1. Sample is added according to the system, and a 20-microliter reaction system of RT-qPCR amplification reaction is as follows: RNA 5. mu.l (0.01 pg-1. mu.g), primer probe (10. mu.M) 2. mu.l, and reaction solution 13. mu.l. Negative controls were added first, followed by the sample and positive controls. The tube caps were closed and centrifuged at 1800rpm for 5 sec. After the sample is added, the residual reagent is immediately put into a refrigerator at the temperature of minus 20 ℃ for storage.
2. And putting the reaction tubes into a PCR instrument in sequence, and carrying out reaction under the following conditions, wherein FAM channels and HEX channels are simultaneously selected by the instrument channels for carrying out experiments. The reaction volume was 20 ul. The qPCR amplification parameters were: keeping at 50 deg.C for 5min and 94 deg.C for 2 min; denaturation at 94 ℃ for 5s and annealing at 55 ℃ for 10s for 40 cycles.
3. Judging the identification result: if the Ct values detected by the FAM channel and the HEX channel are both less than 37, the nucleic acid is judged to be positive by the novel coronavirus (SARS-CoV-2); if the Ct values detected by the FAM channel and the HEX channel have no numerical value or are more than 40, and the detection result of the positive control is positive, the result is judged to be negative by the novel coronavirus (SARS-CoV-2) nucleic acid; if the CT value of the sample to be detected is between 37 and 40, the sample is recommended to be detected again after the nucleic acid is extracted again. If the repeated detection result shows that the Ct value is less than 40 and the amplification curve has obvious peak, the nucleic acid is judged to be positive by the novel coronavirus (SARS-CoV-2); otherwise, the result is negative.
(V) reaction system sensitivity test: when the concentration of the artificially synthesized nucleic acid with positive SARS-CoV-2 is gradually reduced from 1/10 to 1/1000000 by a 10-fold dilution gradient, the cycle number (Ct) of the RFU result reaching the threshold value is less than 35. Meanwhile, under the basic condition that the initial value of the positive control is set to be 9-10ng/ul, the concentration dilution ratio reaches 1000000 times, the result that the cycle number (Ct) is lower than 37 is presented, the lowest detection value of the positive virus can reach 1/1000000, and the method has good sensitivity.
The RT-qPCR technology for detecting the novel coronavirus adopts the RNA reverse transcription and amplification reaction solution with high sensitivity and good stability, all reactions are integrated in one tube, so that the sample processing error is reduced to the maximum extent, the setting time is shortened, and the RNA target can be amplified sensitively and stably; the specific TaqMan probe realizes the standardization of quantitative fluorescent signals, can quickly and simply set reaction, and obtains a detection result within 1 hour. The technical characteristics of saving time and improving the operation efficiency of the laboratory; meanwhile, the method has high sensitivity, economy and high cost performance.
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Claims (3)
1. A method for detecting novel coronavirus by adopting RT-qPCR technology is characterized in that the kit comprises a positive control, a negative control, a primer probe mixed solution and a reaction solution,
1) designing and synthesizing primers and probes; the upstream primer aiming at the N gene is 5'-GGGAGCCTTGAATACACCAAAA-3', the downstream primer is 5'-TGTAGCACGATTGCAGCATTG-3', and the probe primer is 5 '-HEX-AYCACATTGGCACCCGCAATCCTG-BHQ 1-3'; aiming at the orf1a gene, the upstream primer is 5 '-GTGARATGGTCATGTGTGGCGG-3', the downstream primer is 5 '-CARATGTTAAASACACTATTAGCATA-3', and the probe primer is 5 '-FAM-AYCACATTGGCACCCGCAATCCTG-BHQ 2-3'; 2) preparing a reaction system comprising a reaction solution and a probe primer; the reaction solution comprises the following components, magnesium ion concentration is 0.1-10 mu M, ammonium sulfate is 0.1-0.1M, 0.1-10% Tween 20, KCl concentration is 1-1M, reverse transcriptase and DNA high fidelity polymerase; 3) and (3) carrying out RT-qPCR reaction by using the nucleic acid or the recombinant plasmid of the sample to be detected as a template.
2. The method according to claim 1, wherein 1) the final concentration of the primer is 0.01. mu.M-50. mu.M.
3. The method according to claim 1, wherein 1) the final concentration of the probe is 0.01. mu.M to 50. mu.M.
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| CN111676315A (en) * | 2020-04-21 | 2020-09-18 | 中国贵州茅台酒厂(集团)有限责任公司职工医院 | Primer and probe for detecting novel coronavirus ORF1ab gene, kit and method thereof |
| CN112301161A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Primer group and kit for detecting novel coronavirus |
| WO2022079224A1 (en) * | 2020-10-15 | 2022-04-21 | Institut Pasteur | Methods and kits for detecting sars-cov-2 |
| CN112522445A (en) * | 2020-12-23 | 2021-03-19 | 中国科学院上海微系统与信息技术研究所 | Primer-probe combination, kit and method for detecting novel coronavirus |
| CN112730340A (en) * | 2021-01-19 | 2021-04-30 | 西北大学 | Optical fiber sensor for quickly detecting novel coronavirus SARS-CoV-2 |
| CN112730340B (en) * | 2021-01-19 | 2022-03-01 | 西北大学 | Optical fiber sensor for quickly detecting novel coronavirus SARS-CoV-2 |
| CN113005226A (en) * | 2021-02-07 | 2021-06-22 | 利多(香港)有限公司 | Oligonucleotide and kit for detecting SARS-CoV-2 |
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