CN112159868A - Novel coronavirus fluorescence qRT-PCR method rapid detection system - Google Patents
Novel coronavirus fluorescence qRT-PCR method rapid detection system Download PDFInfo
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Abstract
The invention relates to a novel coronavirus fluorescence qRT-PCR method rapid detection system, which realizes the optimization of an amplification system and an amplification program by optimizing a primer probe targeting a novel coronavirus ORF1ab gene and an N gene, enzyme mixed liquor (reverse transcriptase, DNA polymerase and UNG enzyme) of the amplification system, magnesium ion concentration and the like, further shortens the amplification time to within 30min, greatly shortens the detection time, and simultaneously ensures the sensitivity, specificity, repeatability and accuracy of the novel coronavirus detection.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a novel rapid detection system for coronavirus by a fluorescence qRT-PCR method.
Background
2019 the new type coronavirus (SARS-CoV-2) belongs to the genus coronavirus B, and its gene characteristics are obviously different from SARS-CoV and MERS-CoV. Pneumonia caused by SARS-CoV-2 infection is mainly spread by droplet and contact, and is common and susceptible to people. The general symptoms of SARS-CoV-2 infection are: fever, hypodynamia, dry cough, gradually dyspnea, slight symptom of some patients, even no obvious fever. The severe symptoms are: acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis, and procoagulant dysfunction. In addition to the above symptoms, there is also a possibility of having the following atypical symptoms: such as mild anorexia, asthenia, nausea, emesis, diarrhea, headache, etc. From the current accepted cases, the prognosis is good for most patients, and the disease is critical for a few patients, even leading to death in conjunction with other basic diseases. As SARS-CoV-2 has fast spreading property and is easy to cause large-scale epidemic, the method can detect the novel coronavirus timely and accurately and has important significance for epidemic situation monitoring and prevention.
At present, nucleic acid detection kits for detecting SARS-CoV-2 by qRT-PCR are sold on the market, but the detection amplification time is not less than 60 min. The detection methods of the rapid nucleic acid detection kit sold in the market (30min) are all constant-temperature amplification methods, and the sensitivity and the specificity can not reach qRT-PCR detection methods. Therefore, there is an urgent need for a kit capable of rapidly and accurately detecting a novel coronavirus while achieving good sensitivity and specificity.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a novel coronavirus fluorescence qRT-PCR method rapid detection system, the amplification time is within half an hour, but the sensitivity and specificity of SARS-CoV-2 detection both reach the detection level of the standard qRT-PCR sold on the market.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
a primer probe composition comprising a primer probe set selected from the group consisting of:
the primer probe group A comprises a forward primer shown in SEQ ID NO.1, a reverse primer shown in SEQ ID NO.2 and a probe shown in SEQ ID NO. 3;
a primer probe group B for detecting a novel coronavirus N gene, wherein the primer probe group B comprises a forward primer shown by SEQ ID NO.4, a reverse primer shown by SEQ ID NO.5 and a probe shown by SEQ ID NO. 6;
and the primer probe group C comprises a forward primer shown by SEQ ID NO.7, a reverse primer shown by SEQ ID NO.8 and a probe shown by SEQ ID NO. 9.
A kit for the detection of a novel coronavirus comprising a primer probe composition as described above.
Further, the final concentration of the primers of the primer probe sets A and B in the RT-PCR reaction system is 0.8 μ M, the final concentration of the probes of the primer probe sets A and B in the RT-PCR reaction system is 0.4 μ M, the final concentration of the primer of the probe set C in the RT-PCR reaction system is 0.4 μ M, and the final concentration of the primer of the probe set C in the RT-PCR reaction system is 0.2 μ M; in the RT-PCR (reverse Transcription PCR) reaction system, namely a reverse Transcription PCR reaction system, firstly an RNA chain is reversely transcribed into cDNA under the action of reverse transcriptase in the RT-PCR reaction, and then the cDNA is taken as a template to amplify and synthesize a target fragment under the action of DNA polymerase.
Further, the kit also comprises Tris buffer solution, magnesium ions, dA/G/C/UTPs, DNA polymerase, reverse transcriptase, Anti-Taq, BSA, UNG enzyme and RNase.
Further, the final concentration of the reverse transcriptase is preferably 0.4U/. mu.L, and the final concentration of the magnesium ion is preferably 10 mM; further preferably, the reverse transcriptase is a reverse transcriptase having high efficiency and good stability, such asUsing a mixture from ThermoFisher
III Reverse Transcriptase.
Further, the final concentrations of the primers of the primer probe sets A and B are both 0.8. mu.M, the final concentrations of the primers of the primer probe sets A and B are both 0.4. mu.M, the final concentrations of the primers of the primer probe set C are both 0.4. mu.M, the final concentrations of the probes of the primer probe set C are both 0.2. mu.M, the final concentration of magnesium ions is 10mM, the final concentration of dATP is 0.4mM, the final concentration of dCTP is 0.4mM, the final concentration of dGTP is 0.4mM, the final concentration of dUTP is 0.8mM, the final concentration of DNA polymerase is 0.2U/. mu.L, the final concentration of reverse transcriptase is 0.4U/. mu.L, the final concentration of Anti-Taq is 0.2U/. mu.L, the final concentration of BSA is 0.26%, the final concentration of UNG enzyme is 0.004U/. mu.L, and the final concentration of RNase is 0.32U/. mu.L.
Further, the kit also comprises a positive control substance and a negative control substance.
Further, the sample of the novel coronavirus is derived from a human nasopharynx swab, a sputum sample, an alveolar lavage fluid sample, a blood sample, a stool sample, or an environmental sample.
Use of a primer probe composition as described above for the preparation of a kit for the detection of a novel coronavirus by the method comprising:
collecting a sample, extracting novel coronavirus nucleic acid, amplifying the nucleic acid and judging a result;
the result judgment comprises setting a threshold value at an exponential amplification stage on an amplification curve obtained in the nucleic acid amplification step to obtain a corresponding Ct value, and judging a novel coronavirus nucleic acid detection result according to the Ct value;
wherein the judgment of the detection result of the novel coronavirus nucleic acid is the comprehensive judgment of two Ct values obtained by the nucleic acid amplification of two target genes aiming at the novel coronavirus in the same nucleic acid amplification reaction system.
An RT-PCR reaction system for detecting novel coronavirus comprises the primer probe composition, and also comprises Tris buffer solution, magnesium ions, dA/G/C/UTPs, DNA polymerase, reverse transcriptase, Anti-Taq, BSA, UNG enzyme and RNase.
Further, the final concentration of the primers of the primer probe sets A and B in the RT-PCR reaction system is 0.8 μ M, the final concentration of the probes of the primer probe sets A and B in the RT-PCR reaction system is 0.4 μ M, the final concentration of the primer of the probe set C in the RT-PCR reaction system is 0.4 μ M, and the final concentration of the primer of the probe set C in the RT-PCR reaction system is 0.2 μ M.
Further, the final concentration of magnesium ions is 10mM, and the final concentration of reverse transcriptase is 0.4U/. mu.L.
Further, the final concentrations of the primers of the primer probe sets A and B are both 0.8. mu.M, the final concentrations of the primers of the primer probe sets A and B are both 0.4. mu.M, the final concentrations of the primers of the primer probe set C are both 0.4. mu.M, the final concentration of the magnesium ion is 10mM, the final concentration of the dATP is 0.4mM, the final concentration of the dCTP is 0.4mM, the final concentration of the dGTP is 0.4mM, the final concentration of the dUTP is 0.4mM, the final concentration of the DNA polymerase is 0.2U/μ L, the final concentration of the reverse transcriptase is 0.4U/μ L, the final concentration of the Anti-Taq is 0.2U/μ L, the final concentration of the reverse transcriptase is 0.26%, the final concentration of the UNG enzyme is 0.004U/μ L, and the final concentration of the RNase is 0.32U/μ L.
A novel coronavirus nucleic acid amplification method, comprising:
s1, providing any RT-PCR reaction system as described above, and carrying out reverse transcription reaction;
s2, carrying out the reactions of pre-denaturation, annealing and chain extension of nucleic acid.
Further, the reverse transcription reaction time is 2 min; the pre-denaturation time is 2 s; the denaturation time was 1 s.
TABLE 1SEQ ID NO. 1-9 sequence information table
The invention has the beneficial effects that:
by adopting the novel coronavirus fluorescence qRT-PCR rapid detection system, the optimization of an amplification system and an amplification program is realized by optimizing a primer probe targeting a novel coronavirus ORF1ab gene and an N gene and the concentration of corresponding components of an enzyme mixed solution (reverse transcriptase, DNA polymerase and UNG enzyme) and magnesium ions of the amplification system, so that the amplification time is shortened to be within 30min, the detection time is greatly shortened, and the sensitivity, the specificity, the repeatability and the accuracy of the novel coronavirus detection are ensured.
Drawings
FIG. 1 provides a schematic illustration of an amplification curve analysis according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing the detection sensitivity of the N gene in the embodiment of the present invention.
FIG. 3 is a schematic diagram showing the detection sensitivity of ORF1ab gene in the example of the present invention.
FIG. 4 is a schematic diagram showing the detection specificity of ORF1ab gene in the examples of the present invention.
FIG. 5 is a schematic diagram of the specificity of N gene detection provided in the embodiment of the present invention.
FIG. 6 is a schematic diagram of the accuracy of the kit for detecting the novel coronavirus culture provided by the embodiment of the invention.
FIG. 7 is a schematic diagram of the accuracy of the kit for detecting a novel coronavirus throat swab sample according to the embodiment of the invention.
FIG. 8 is a schematic diagram of the accuracy of the kit for detecting the novel coronavirus culture provided by the embodiment of the invention.
FIG. 9 is a schematic diagram of the accuracy of the kit for detecting a novel coronavirus throat swab sample according to the embodiment of the invention.
FIG. 10 is a schematic diagram of the accuracy of the kit for detecting a novel coronavirus throat swab sample according to the embodiment of the invention.
FIG. 11 is a schematic diagram of the accuracy of the kit for detecting a novel coronavirus throat swab sample according to the embodiment of the invention.
FIG. 12 is a schematic diagram showing the reproducibility of ORF1ab gene detection in the examples of the present invention.
FIG. 13 is a schematic diagram showing the reproducibility of the detection of N gene in the embodiment of the present invention.
FIG. 14 is a schematic diagram of the detection of the laboratory quality assessment sample 202001 provided in the kit according to the embodiment of the present invention.
FIG. 15 is a schematic diagram of the detection of the laboratory quality assessment sample 202002 provided in the kit according to the embodiment of the present invention.
FIG. 16 is a schematic diagram of the detection of the laboratory quality assessment sample 202003 provided in the kit according to the embodiment of the present invention.
FIG. 17 is a schematic diagram of the detection of the laboratory quality assessment sample 202004 of the kit provided by the embodiment of the invention.
FIG. 18 is a schematic diagram of the detection of the laboratory quality assessment sample 202005 of the kit provided by the embodiment of the invention.
FIG. 19 is a schematic diagram of the detection of the laboratory quality assessment sample 202006 provided in the kit according to the embodiment of the present invention.
FIG. 20 is a schematic diagram of a laboratory quality assessment sample 202007 detection kit provided by the embodiment of the invention.
Fig. 21 is a schematic diagram of the detection of a laboratory quality assessment sample 202008 with the kit according to the embodiment of the present invention.
FIG. 22 is a schematic diagram of the detection of the laboratory quality assessment sample 202009 provided in the kit according to the embodiment of the present invention.
FIG. 23 is a schematic diagram of the detection of the laboratory quality assessment sample 202010 provided in the kit according to the embodiment of the present invention.
Fig. 24 is a schematic view of the verification of the interference experiment of the kit provided by the embodiment of the present invention.
FIG. 25 is a schematic diagram of detection on day 0 of an accelerated stability test provided by the kit according to the embodiment of the present invention.
FIG. 26 is a schematic view of detection on day 1 of an accelerated stability test provided by an embodiment of the present invention.
FIG. 27 is a schematic diagram of detection on day 4 of an accelerated stability test provided by the kit according to the embodiment of the present invention.
FIG. 28 is a schematic diagram of detection of the kit on day 9 in the accelerated stability test provided in the embodiments of the present invention.
Detailed Description
For a clearer understanding of the contents of the present invention, reference will be made to the accompanying drawings and examples.
Example 1 primer probes, reagents and detection methods
1.1 primer Probe sequences
Selecting ORF1ab gene of novel coronavirus SARS-CoV-2 as amplification target region, designing specific primer and fluorescent probe, labeling the probe with FAM, and detecting whether the sample contains novel coronavirus RNA. Selecting a novel coronavirus N gene, designing a specific primer and a fluorescent probe, marking the probe by ROX, and detecting whether a sample contains novel coronavirus RNA. And (3) selecting other species sequences of the non-human genome non-kit detection target to design an internal standard primer probe, marking the probe by VIC, and adding the internal standard primer probe into a reaction system to monitor the whole experimental process.
1.2 reagents required for the detection of the novel coronavirus SARS-CoV-2 are shown in Table 2, and the concentrations of the respective components are shown in Table 3.
TABLE 2 composition of reagents
| Name of reagent | The main components |
| Negative control | Nuclease-free water |
| Positive control | Novel coronavirus plasmid |
| Internal standard | MS2 RNA pseudovirus |
| RT-PCR reaction solution | Tris, magnesium ion, dA/G/C/UTPs, Anti-Taq, BSA, RNasen |
| Enzyme mixingArticle (A) | DNA polymerase, reverse transcriptase, UNG enzyme |
| Novel coronavirus primer probe | Oligonucleotides |
TABLE 3 concentrations of the components
1.3 sample treatment
The nucleic acid extraction kit is used for extracting and purifying sample nucleic acid, and the nucleic acid extraction is not needed for positive control and negative control, and the nucleic acid detection is directly carried out.
1.4 sample application
Adding processed negative control, positive control and sample nucleic acid to be detected into prepared PCR reaction tube (plate) respectively by 12.5 μ L, covering tube cover (sealing membrane), centrifuging for a short time, and transferring to amplification detection zone.
1.5 amplification assay
(1) PCR reaction solution was prepared according to the number of samples to be amplified (including positive control and negative control) in the above ratio of 3, and dispensed into PCR reaction tubes (plates) in a volume of 12.5. mu.L.
(2) Adding 12.5 mul RNA template, negative control and positive control, covering tube cover (sealing film if PCR reaction plate), laying aside at room temperature (15-25 deg.C) for 5 min, transferring to amplification detection zone, and putting PCR reaction tube (plate) into Thermo Q5 fluorescence PCR amplification instrument for amplification detection.
(3) Setting amplification detection parameters:
(4) amplification reaction and detection
Amplification reaction and detection were performed as described above, and the Ct value of the sample was determined.
(5) Reference value (Ct value)
Through the detection of the negative sample and the sample close to the lowest detection limit, the reference value of the FAM channel, the reference value of the ROX channel and the reference value of the VIC channel of the kit are determined to be 39.5, 39.5 and 38.0 respectively.
(6) Determination of detection result
The test results of the samples to be tested were determined according to table 4:
TABLE 4 determination method of novel coronavirus detection results
Example 2 detection sensitivity
The primer probe combination, the reagent and the nucleic acid amplification detection method in the example 1 are used for verifying the sensitivity, the national standard 2019-nCoV nucleic acid detection reagent national reference (370099-5copies/mL, using a combination of digital PCR methods. S was diluted 1:3 times (2 parts water +1 part sample) with RNA/DNase-free deionized water, and then labeled S1 to S10 at 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, 1:6561, 1:19683, 1:59049, and 1:177147, respectively, and nucleic acid was extracted according to the kit instructions and then detected. According to the detection result after the gradient dilution, the detection sensitivity of the kit to SARS-CoV-2 is 0.046 copies/. mu.L, as shown in FIG. 2 and FIG. 3.
Example 3 negative match Rate (specificity)
Specificity was verified using the primer probe combination, reagents and nucleic acid amplification detection method of example 1.
In order to prove the cross reactivity of the kit, the specificity of a detection system is verified by using the reagent and the nucleic acid amplification detection method, and a national standard product 2019-nCoV nucleic acid detection reagent national reference product (370099-.
Including the following pathogens: human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1, SARS coronavirus, MERS coronavirus, avian influenza virus H5N1, influenza B Virus (BV), influenza A H1N1(2009) virus, influenza A H1N1 virus, influenza A H3N2 virus, EB virus, meningococcus, Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, rubella virus, mumps virus, respiratory adenovirus type 3, respiratory adenovirus type 7, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus type A, respiratory syncytial virus type B, parainfluenza virus type 2, Legionella pneumophila, Bordetella pertussis and human genomic DNA were detected separately.
As can be seen from the reaction results of FIG. 4 and FIG. 5, there is no cross-reactivity between 28 pathogens and human genome DNA, and the detection results are negative, so that the specificity is good.
Example 4 Positive compliance-national Standard (accuracy)
The primer probe combination, reagents and nucleic acid amplification detection method of example 1 were used to verify accuracy.
In order to prove the accuracy of the kit, the detection accuracy of a detection system is verified by using the reagent and the nucleic acid amplification detection method, and a national reference product (370099-.
The positive reference substance comprises 6 detection samples such as a novel coronavirus culture, a throat swab sample and a plasmid (N full-length gene), and the detection results of the kit can be seen from the detection results of fig. 6, 7, 8, 9, 10 and 11, and the accuracy rate of the positive reference substance detected by the kit is 100%.
Example 5 reproducibility test-national Standard
Repetitive detection was performed using the primer probe combination, reagents and nucleic acid amplification detection method of example 1.
The kit is used for carrying out repetitive detection for 10 times, and the detection results are shown in Table 5.
TABLE 5 results of repeated measurements
As can be seen from the detection results of Table 5, FIG. 12 and FIG. 13, the kit has better detection repeatability for the novel coronavirus.
Example 6 laboratory evaluation sample testing
The primer probe combination, the reagent and the nucleic acid amplification detection method in example 1 are used for detecting the laboratory quality evaluation sample.
Sample numbers and concentrations are shown in table 6.
TABLE 6 laboratory quality assessment sample testing
| Sample numbering | SARS-CoV-2 results | Composition (I) | Concentration (copies/mL) |
| 202001 | Negative of | SARS | 1.0×107 |
| 202002 | Positive for | SARS-CoV-2 | 5.5×104 |
| 202003 | Positive for | SARS-CoV-2 | 3.5×106 |
| 202004 | Positive for | SARS-CoV-2 | 1.4×104 |
| 202005 | Positive for | SARS-CoV-2 | 2.2×105 |
| 202006 | Negative of | / | / |
| 202007 | Positive for | SARS-CoV-2 | |
| 202008 | Negative of | / | / |
| 202009 | Positive for | SARS-CoV-2 | 3.5×103 |
| 202010 | Positive for | SARS-CoV-2 | 8.8×105 |
As can be seen from the test results in table 6 and fig. 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, the test coincidence rate of the kit on the laboratory quality evaluation sample is 100%.
Example 7 interfering substances
The detection of interfering substances was carried out by using the primer probe combination, the reagent and the nucleic acid amplification detection method of example 1.
Selecting 4 sampling interference substances: normal human fresh blood, nasal secretions, mucus, mucin, and 24 drugs commonly used in respiratory infections as interfering substances: phenylephrine, oxymetazoline, sodium chloride, beclomethasone, dexamethasone, flunisolide, triamcinolone acetonide, budesonide, mometasone, fluticasone, interferon-alpha, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, abidol, mupirocin, levofloxacin, azithromycin, ceftriaxone, meropenem, tobramycin, and the effects of these interfering substances were studied.
And (3) taking a middle-low concentration simulation sample (pseudovirus + negative sample), taking a sampling interfering substance with the proportion or concentration of 10% of the medical level and the highest concentration of the dosage in the medical instruction as an interfering concentration to be added into the diluted sample, taking an uncontained interfering substance as a control, carrying out negative and positive judgment on the result, and evaluating the influence of each interfering substance on the detection result before and after the addition. As can be seen from the test results in fig. 24, the samples added with 4 sampling interfering substances and 24 drugs do not affect the determination of the negative or positive test results compared with the controls.
Example 8 accelerated stability test
An accelerated stability test was performed using the primer probe combination, the reagent and the nucleic acid amplification detection method of example 1.
And (3) placing the kit in a 37 ℃ incubator, and sequentially testing the medium-concentration samples and the low-concentration samples on the 0 th day, the 1 st day, the 4 th day and the 9 th day according to national standards and industrial standards. As can be seen from the detection results of fig. 25, 26, 27, and 28, the accuracy of the detection result of this kit was not affected by the standing time.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
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Claims (15)
1. A primer probe composition comprising a primer probe set selected from the group consisting of:
the primer probe group A comprises a forward primer shown in SEQ ID NO.1, a reverse primer shown in SEQ ID NO.2 and a probe shown in SEQ ID NO. 3;
a primer probe group B for detecting a novel coronavirus N gene, wherein the primer probe group B comprises a forward primer shown by SEQ ID NO.4, a reverse primer shown by SEQ ID NO.5 and a probe shown by SEQ ID NO. 6;
and the primer probe group C comprises a forward primer shown by SEQ ID NO.7, a reverse primer shown by SEQ ID NO.8 and a probe shown by SEQ ID NO. 9.
2. A kit for the detection of a novel coronavirus, said kit comprising the primer probe composition of claim 1.
3. The kit of claim 2, wherein the final concentration of the primers of the primer probe sets A and B in the RT-PCR reaction system is 0.8 μ M, the final concentration of the probes of the primer probe sets A and B in the RT-PCR reaction system is 0.4 μ M, the final concentration of the primers of the probe set C in the RT-PCR reaction system is 0.4 μ M, and the final concentration of the primers of the probe set C in the RT-PCR reaction system is 0.2 μ M.
4. The kit of claim 2, wherein the kit further comprises Tris buffer, magnesium ions, dA/G/C/UTPs, DNA polymerase, reverse transcriptase, Anti-Taq, BSA, UNG enzyme, RNasin.
5. The kit of claim 4, wherein the final concentration of reverse transcriptase is 0.4U/. mu.L and the final concentration of magnesium ions is 10 mM.
6. The kit of claim 4, wherein the primer final concentrations of the primer probe sets A and B are 0.8 μ M, the primer final concentrations of the primer probe sets A and B are 0.4 μ M, the primer final concentration of the primer probe set C is 0.4 μ M, the probe final concentration of the primer probe set C is 0.2 μ M, the final concentration of magnesium ions is 10mM, the final concentration of dATP is 0.4mM, the final concentration of dCTP is 0.4mM, the final concentration of dGTP is 0.4mM, the final concentration of dUTP is 0.8mM, the final concentration of DNA polymerase is 0.2U/μ L, the final concentration of reverse transcriptase is 0.4U/μ L, the final concentration of Anti-Taq is 0.2U/μ L, the final concentration of BSA is 0.26%, the final concentration of UNG enzyme is 0.004U/μ L, and the final concentration of RNase is 0.32U/μ L.
7. The kit of claim 4, further comprising a positive control and a negative control.
8. The kit of claim 2, wherein the sample of the novel coronavirus is derived from a human nasopharynx swab, a sputum sample, an alveolar lavage sample, a blood sample, a stool sample, or an environmental sample.
9. Use of a primer probe composition according to claim 1 for the preparation of a kit for the detection of a novel coronavirus by a method comprising:
collecting a sample, extracting novel coronavirus nucleic acid, amplifying the nucleic acid and judging a result;
the result judgment comprises setting a threshold value at an exponential amplification stage on an amplification curve obtained in the nucleic acid amplification step to obtain a corresponding Ct value, and judging a novel coronavirus nucleic acid detection result according to the Ct value;
wherein the judgment of the detection result of the novel coronavirus nucleic acid is the comprehensive judgment of two Ct values obtained by the nucleic acid amplification of two target genes aiming at the novel coronavirus in the same nucleic acid amplification reaction system.
10. An RT-PCR reaction system for the detection of a novel coronavirus, comprising the primer probe composition of claim 1, and further comprising Tris buffer, magnesium ions, dA/G/C/UTPs, DNA polymerase, reverse transcriptase, Anti-Taq, BSA, UNG enzyme, RNasin.
11. The RT-PCR reaction system of claim 10, wherein the final concentration of the primers of the primer probe sets A and B in the RT-PCR reaction system is 0.8 μ M, the final concentration of the probes of the primer probe sets A and B in the RT-PCR reaction system is 0.4 μ M, the final concentration of the primers of the primer probe set C in the RT-PCR reaction system is 0.4 μ M, and the final concentration of the primers of the primer probe set C in the RT-PCR reaction system is 0.2 μ M.
12. The RT-PCR reaction system of claim 10, wherein the final concentration of magnesium ions is 10mM and the final concentration of reverse transcriptase is 0.4U/. mu.L.
13. The RT-PCR reaction system according to claim 10, the final concentrations of the primers of the primer probe sets A and B are both 0.8 μ M, the final concentrations of the primers of the primer probe sets A and B are both 0.4 μ M, the final concentrations of the primers of the primer probe set C are both 0.4 μ M, the final concentrations of the probes of the primer probe set C are both 0.2 μ M, the final concentration of magnesium ions is 10mM, the final concentration of dATP is 0.4mM, the final concentration of dCTP is 0.4mM, the final concentration of dGTP is 0.4mM, the final concentration of dUTP is 0.4mM, the final concentration of DNA polymerase is 0.2U/μ L, the final concentration of reverse transcriptase is 0.4U/μ L, the final concentration of AngTaq-Taq is 0.2U/μ L, the final concentration of BSA is 0.26%, the final concentration of UNG enzyme is 0.004U/μ L, and the final concentration of RNase is 0.32U/μ L.
14. A novel coronavirus nucleic acid amplification method, comprising:
s1, providing the RT-PCR reaction system as in any one of the claims 10-13, and carrying out reverse transcription reaction;
s2, carrying out the reactions of pre-denaturation, annealing and chain extension of nucleic acid.
15. The amplification method of claim 14, wherein the reverse transcription reaction time is 2 min; the pre-denaturation time is 2 s; the denaturation time was 1 s.
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