CN111621604A - Novel primer probe composition, kit and method for coronavirus nucleic acid detection - Google Patents

Novel primer probe composition, kit and method for coronavirus nucleic acid detection Download PDF

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CN111621604A
CN111621604A CN202010588373.0A CN202010588373A CN111621604A CN 111621604 A CN111621604 A CN 111621604A CN 202010588373 A CN202010588373 A CN 202010588373A CN 111621604 A CN111621604 A CN 111621604A
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方耀东
陈林
李明阳
韩丽
张文学
古迪
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Shanghai Chromysky Medical Research Co ltd
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Abstract

The invention relates to the technical field of biotechnology and medical inspection, and provides a novel primer probe composition, a kit and a method for detecting coronavirus nucleic acid for solving the problems of low accuracy, poor specificity and low sensitivity of the traditional virus nucleic acid detection method, wherein the novel primer probe composition for detecting coronavirus nucleic acid comprises the following components in parts by weight: a first primer probe set comprising a forward primer ORF1ab-F, a probe ORF1ab-P and a reverse primer ORF1 ab-R; a second primer probe set comprising a forward primer N-F, a probe N-P and a reverse primer N-R; and the third primer probe set comprises a forward primer Rpp30-F, a probe Rpp30-P and a reverse primer Rpp 30-R. The primer probe composition avoids mutual interference between a plurality of primer pairs and corresponding detection probes through the ingenious design of the amplification primer pairs and the detection probes; the kit has a simple structure, and can monitor the collection, transportation and extraction processes of the sample to be detected through the internal standard, thereby avoiding false negative of the detection result.

Description

Novel primer probe composition, kit and method for coronavirus nucleic acid detection
Technical Field
The invention relates to the technical field of biotechnology and medical inspection, in particular to a novel primer probe composition, a kit and a method for detecting coronavirus nucleic acid.
Background
The novel coronavirus (COVID-19) is a positive-strand single-stranded RNA coronavirus with an envelope, each group of which has a genome length of about 30000 nucleotides and belongs to the coronavirus lineage B (Betaconovir Linage B, Sarbecovirus) together with SARS-CoV. Similar to SARS-CoV, COVID-19 can bind via human ACE2 as a receptor, enter the lung through airway epithelial cells to perform the replication process, and the main target is also the same T lymphocyte as SARS-CoV. It is primarily transmitted by unprotected intimate contact and spray with infected persons, and possibly also by aerosol. The basic clinical symptoms are fever, dry cough and hypodynamia as main manifestations. A few patients have nasal obstruction, watery nasal discharge, pharyngalgia, myalgia and diarrhea. Severe patients are often found to develop dyspnea and/or hypoxemia after one week of onset, and severe patients may rapidly progress to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis and coagulation dysfunction, and multiple organ failure. Based on the current epidemiological investigation, the incubation period is 1 to 14 days, and is mostly 3 to 7 days. All the viruses are susceptible and have no specific medicine temporarily. Because the high lethality rate is similar to the symptoms of cold and pneumonia caused by other viruses, and the prevention, control and treatment of diseases are not facilitated, the design of a rapid, accurate and high-flux diagnostic kit specially used for differential diagnosis of the virus is of great social significance.
The novel coronavirus is RNA virus, so that the extraction and storage processes of the coronavirus are easy to destroy virus nucleic acid, and false negative is caused in detection. Furthermore, due to the high variability of the virus, false negatives can result if primer probes are not designed satisfactorily. The existing virus nucleic acid detection methods mainly comprise the following methods:
the fluorescent quantitative PCR method is mainly used for in vitro qualitative detection of viral nucleic acid, and the samples mainly comprise nasopharyngeal swab, alveolar lavage fluid, sputum, anal swab, excrement and the like. The detection result is generally confirmed for 4-5 hours from sample extraction. The sensitivity is high, and the false positive and false negative rates are relatively low; the experts have consensus; the window period is relatively short; directly aims at the target virus and can judge the degree of virus infection and the effect after the administration through relative abundance.
Compared with a PCR method, the sequencing method has longer time, higher requirements on laboratories and specialties, higher detection cost and more benefit for variation research of strains. The isothermal amplification method is slightly shorter than PCR in detection time and high in sensitivity, but has higher design requirement on primers, the development and matching of the whole industry are not as mature as PCR, professional personnel and equipment are needed, and the false positive rate is higher. The main detection object of the colloidal gold and the chemiluminescence method is a specific antibody in blood, and the method has the characteristics of short detection time, high speed, low detection cost and long detection window period in clinic. The colloidal gold method has the advantages of no dependence on professionals and equipment, simple operation, and the defects of poor specificity, low sensitivity, high false negative rate and high false positive rate. The chemiluminescence method has higher sensitivity compared with the colloidal gold method, but requires special equipment and personnel.
Although the fluorescent quantitative PCR method has the advantages of high speed, high flux, strong universality and matched maturity, the fluorescent quantitative PCR method has the disadvantages of high requirement on the professional of personnel, need of professional equipment and laboratories, high detection cost, easy damage to virus nucleic acid due to long-distance transportation of samples and the like.
Disclosure of Invention
In order to overcome the problems of low accuracy, poor specificity and low sensitivity of the traditional virus nucleic acid detection method, the invention provides a novel primer probe composition for coronavirus nucleic acid detection, which has high accuracy, good specificity and high sensitivity, and avoids mutual interference between a plurality of primer pairs and corresponding detection probes.
The invention also provides a novel coronavirus nucleic acid detection kit.
The invention also provides a novel coronavirus nucleic acid detection method which is high in specificity, accuracy and sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
as shown in table 1, a novel coronavirus nucleic acid detection primer probe composition comprises:
the first primer probe group comprises a forward primer ORF1ab-F, a probe ORF1ab-P and a reverse primer ORF1ab-R, and the nucleotide sequences of the first primer probe group are respectively shown as SEQ ID Nos. 1-3; the first primer probe group takes ORF1ab gene of the novel coronavirus as a detection target point;
a second primer probe group, which comprises a forward primer N-F, a probe N-P and a reverse primer N-R, wherein the nucleotide sequences of the second primer probe group are respectively shown as SEQ ID Nos. 4-6; the second primer probe group takes the N gene of the novel coronavirus as a detection target;
a third primer probe set which comprises a forward primer Rpp30-F, a probe Rpp30-P and a reverse primer Rpp30-R, wherein the nucleotide sequences of the third primer probe set are respectively shown as SEQ ID Nos. 7-9; the third primer probe group takes mRNA of human RPP30 gene as a detection target.
The nucleotide sequence is not limited to the sequence shown in SEQ ID No. 1-9, and also comprises a reverse complementary sequence and a homologous similar sequence thereof.
TABLE 1 primer Probe compositions
Figure BDA0002554607850000031
The novel coronavirus nucleic acid detection primer probe composition provided by the invention is based on the combination of a primer probe of novel coronavirus ORF1ab and N genes and a human-derived sample housekeeping gene Rpp30, and mutual interference between a plurality of primer pairs and corresponding detection probes is avoided through the ingenious design of amplification primer pairs and detection probes; and proved that when the primer probe combination is used for detecting the novel coronavirus, the specificity is good, and the sensitivity is high.
As shown in Table 2, the primer probes of the novel coronavirus ORF1ab and N genes are designed according to the gene sequences SEQ No.10 and SEQ No.11 of the novel coronavirus COVID-19 published by the national center for disease prevention and control in China; the gene sequence of the humanized sample housekeeping gene Rpp30 is shown in SEQ No.12, and the humanized sample housekeeping gene is ubiquitous in a sample, high in specificity and highly conserved.
TABLE 2 target sequences
Figure BDA0002554607850000032
The design idea of the novel coronavirus nucleic acid detection primer probe composition provided by the invention is as follows:
in order to ensure the accuracy of the detection result of the novel coronavirus, two genes of SEQ ID No10 and SEQ ID No11 in a virus genome are selected as target sequences, specific primer probes (SEQ ID No. 1-6) are designed for amplification, a fluorescence-free amplification signal of human genome DNA or other pathogen genomes is detected, and a fluorescence amplification signal of the novel coronavirus genome is detected.
In order to eliminate false negative, false positive, pollution and extraction efficiency, human genome mRNA (SEQ ID No12) is selected as a target sequence, specific primer probes (SEQ ID Nos. 7-9) are designed for amplification, a pathogen genome fluorescence-free amplification signal is detected, and a human RPP30 gene mRNA fluorescence amplification signal is detected.
Preferably, the 5 'end of the probe ORF1ab-P, the probe N-P and the probe Rpp30-P is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group.
Preferably, the fluorescent group is selected from at least 1 of FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red, and CY 3. The fluorescent group labeled by each probe is different.
Preferably, the fluorescence quenching group is at least 1 selected from MGB, BHQ-1, BHQ-2, BHQ-3 and TAMRA. A novel coronavirus nucleic acid detection kit of the primer probe composition comprises a primer probe composition container, an internal and external quality control container, a reverse transcription reaction and PCR amplification reaction container, wherein the primer probe composition container contains primer probe dry powder or solution with a nucleotide sequence shown as SEQ ID No. 1-9; the inner and outer quality control containers contain dry powder or solution with nucleotide sequences shown as SEQ ID No. 10-12; the reverse transcription reaction and PCR amplification reaction vessel comprises reverse transcriptase, DNA polymerase, auxiliary reinforcing agent and buffer solution.
The novel coronavirus nucleic acid detection kit provided by the invention is characterized in that a protein subunit Rpp30 of RNase P is used as an endogenous internal standard, a CY5 fluorescein is adopted for marking, and the internal standard is used for monitoring the collection, transportation and extraction processes of a sample to be detected, so that false negative of a detection result is avoided.
Preferably, the pH value of the buffer solution is controlled to be 7.5-9.5 (25 ℃).
Preferably, the novel coronavirus nucleic acid detection kit further comprises a positive control group and a negative control group, wherein the positive control group is novel coronavirus COVID-19 nucleic acid or pseudovirion; the negative control group is human source sample housekeeping gene pseudovirion.
A method for novel coronavirus nucleic acid detection using the primer probe composition described above for non-diagnostic purposes, comprising the steps of:
(1) extracting and purifying nucleic acid of the novel coronavirus sample to obtain a nucleic acid extracting solution;
(2) establishing a fluorescent quantitative PCR system containing the primer probe composition by taking the nucleic acid extracting solution as a template;
(3) and detecting the PCR reaction mixed solution containing the template by a fluorescent quantitative PCR detection system, and judging whether the patient is infected with the novel coronavirus according to the obtained different fluorescent signal data. When the probe is complete, no fluorescence can be detected, in the PCR extension process, the DNA polymerase hydrolyzes the probe and then emits a fluorescence signal, and the fluorescence increase value is positively correlated with the amount of PCR products.
The detection principle of the invention is as follows: the ORF1ab/N gene of the novel coronavirus was selected as the target for detection. A set of primer probes is designed according to the detection target, wherein the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a fluorescent quenching group. When the probe remains intact, the fluorescence quencher can significantly reduce the fluorescence emitted by the fluorophore via Fluorescence Resonance Energy Transfer (FRET). During PCR amplification, the primer and the probe are combined to the template at the same time, and the combination position of the probe is positioned between the upstream primer and the downstream primer. When amplification extends to the position where the probe binds, Taq enzyme cleaves the fluorescent molecule attached to the 5 'end of the probe from the probe using 5' exonuclease activity, thereby causing it to fluoresce. The number of detected fluorescent molecules is proportional to the number of PCR products, and therefore, the number of initial DNA templates can be calculated according to the fluorescence intensity in the PCR reaction system. The fluorescent quantitative PCR instrument automatically draws a real-time amplification curve according to the detected fluorescent signal, thereby realizing the qualitative detection of the novel coronavirus at the nucleic acid level.
Preferably, the fluorescent quantitative PCR system comprises a primer probe mixture of the novel coronavirus ORF1ab, the N gene and the human-derived sample housekeeping gene.
Preferably, the fluorescent quantitative PCR system further comprises reverse transcriptase, DNA polymerase, dNTP and Mg2+、ROXDye。
Preferably, the PCR system further comprises at least one of a PCR buffer system, a PCR enhancer and a PCR stabilizer, and the PCR buffer system comprises Tris (10 mM-500 mM), KCl (10 mM-500 mM) and (NH)4)2SO4(10 mM-500 mM), glycerol (1% -50%), BSA (0.001 mg/mL-1 mg/mL), Tween 20 (0.1% -10%), dithiothreitol (1 mM/mL-100 mM/mL), betaine (0.1M-3M) and DMSO (0.1% -10%).
Preferably, the human derived sample housekeeping gene is selected from at least 1 of the protein subunits Rpp 20, Rpp30, Rpp40, GAPDH and β -actin of Rnase P.
Therefore, the invention has the following beneficial effects:
(1) the novel coronavirus nucleic acid detection primer probe composition avoids mutual interference between a plurality of primer pairs and corresponding detection probes through the ingenious design of the amplification primer pairs and the detection probes;
(2) the novel coronavirus nucleic acid detection kit provided by the invention is characterized in that a protein subunit Rpp30 of RNase P is used as an endogenous internal standard, a CY5 fluorescein is adopted for marking, and the internal standard is used for monitoring the collection, transportation and extraction processes of a sample to be detected, so that false negative of a detection result is avoided;
(3) the novel coronavirus nucleic acid detection method disclosed by the invention realizes qualitative detection of the novel coronavirus on a nucleic acid level based on a PCR reaction system established by the primer probe composition, and the detection method is high in accuracy, good in specificity and high in sensitivity.
Drawings
FIG. 1 is a comparison graph of the fluorescent quantitative PCR amplification curve of the sensitivity of the novel coronavirus nucleic acid detection kit.
FIG. 2 is a comparison graph of fluorescent quantitative PCR amplification curves of the lowest detection limit of the novel coronavirus nucleic acid detection kit.
FIG. 3 is a comparison of fluorescent quantitative PCR amplification curves of clinical samples of the novel coronavirus nucleic acid detection kit.
Detailed Description
The technical solution of the present invention is further specifically described below by using specific embodiments and with reference to the accompanying drawings.
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
In order to ensure the accuracy of the detection result of the novel coronavirus, two genes of SEQ ID No10 and SEQ ID No11 in a virus genome are selected as target sequences, specific primer probes (SEQ ID No. 1-6) are designed for amplification, a fluorescence-free amplification signal of human genome DNA or other pathogen genomes is detected, and a fluorescence amplification signal of the novel coronavirus genome is detected.
In order to eliminate false negative, false positive, pollution and extraction efficiency, human genome mRNA (SEQ ID No12) is selected as a target sequence, specific primer probes (SEQ ID Nos. 7-9) are designed for amplification, a pathogen genome fluorescence-free amplification signal is detected, and a human RPP30 gene mRNA fluorescence amplification signal is detected.
A novel coronavirus nucleic acid detection primer probe composition comprising:
the first primer probe group comprises a forward primer ORF1ab-F, a probe ORF1ab-P and a reverse primer ORF1ab-R, and the nucleotide sequences of the first primer probe group are respectively shown as SEQ ID Nos. 1-3; wherein, the 5 'end of the probe ORF1ab-P is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BHQ-1;
a second primer probe group, which comprises a forward primer N-F, a probe N-P and a reverse primer N-R, wherein the nucleotide sequences of the second primer probe group are respectively shown as SEQ ID Nos. 4-6; wherein, the 5 'end of the probe N-P is marked with a fluorescent group VIC, and the 3' end is marked with a fluorescence quenching group BHQ-2;
a third primer probe set which comprises a forward primer Rpp30-F, a probe Rpp30-P and a reverse primer Rpp30-R, wherein the nucleotide sequences of the third primer probe set are respectively shown as SEQ ID Nos. 7-9; wherein, the 5 'end of the probe Rpp30-P is marked with a fluorescence group CY5, and the 3' end is marked with a fluorescence quenching group BHQ-2.
A novel coronavirus nucleic acid detection kit containing the primer probe composition comprises a primer probe composition container, an internal and external quality control container, a reverse transcription reaction and PCR amplification reaction container, wherein the primer probe composition container contains primer probe dry powder or solution with a nucleotide sequence shown as SEQ ID No. 1-9; the inner and outer quality control containers contain dry powder or solution with nucleotide sequences shown as SEQ ID No. 10-12; the reverse transcription reaction and PCR amplification reaction vessel comprises reverse transcriptase, DNA polymerase, auxiliary reinforcing agent and buffer solution.
Specifically, this example provides a kit as shown in table 3:
TABLE 3 kit
Serial number Name of reagent Principal Components
1 CVD main reaction liquid Reverse transcriptase, DNA polymerase, dNTP, Mg2+, and buffer solution with pH value of 7.5-9.5
2 CVD reaction liquid New coronavirus ORF1ab, N gene, human Rpp30mRNA primer and probe mixed liquor
3 CVD positive control Mixed pseudovirions comprising ORF1ab, N gene
4 CVD negative control RPP30 pseudovirions
The detection stability, sensitivity and accuracy of the novel coronavirus nucleic acid detection kit are judged, and the results are shown in the comparison graphs of fluorescent quantitative PCR amplification curves shown in figures 1-3:
FIG. 1 shows a pseudovirion mixture comprising the ORF1ab and the N gene of a novel coronavirus, which pseudovirion mixture was obtained by using RNase-Free H2O was diluted in 10-fold gradient at 1 × 10 concentration9、1×108、1×107、1×106、1×105、1×104、1×103copies/mL, detection by using the kit, detection resultAll were positive. Coefficient of correlation R2All are larger than 0.999, which indicates that the detection effect of the kit is stable.
FIG. 2 shows the use of RNase-Free H as the nucleic acid of a novel coronavirus positive clinical specimen2Dilution of O to 1 × 103The copies/mL is repeatedly detected for 25 times by adopting the kit, the ORF1ab and N gene detection is positive, the detection rate is 100 percent, and the kit is proved to have high sensitivity.
FIG. 3 shows that the detection results of the nucleic acids of the samples clinically confirmed as positive and negative of the new coronavirus are consistent with the clinical results of the detection by the kit, which indicates that the kit of the present invention has high accuracy.
A method for detecting novel coronavirus nucleic acid by using the primer probe composition comprises the following steps:
(1) extracting and purifying nucleic acid of the novel coronavirus sample to obtain a nucleic acid extracting solution;
sample types include: upper respiratory specimens (including but not limited to pharyngeal swabs, nasal swabs, nasopharyngeal aspirates, profuse sputum); lower respiratory tract specimens (including but not limited to respiratory tract aspirates, bronchial lavage, alveolar lavage, lung biopsy specimens); tissue cultures, serum, virus preservation solutions, and the like;
collecting samples: collecting samples according to the requirements of a manual for collecting microorganism samples;
sample preservation: the collected specimen should be sent for inspection in time, and the specimen should be stored at 4 ℃ within 24 hours; the product can be preserved at-70 deg.C for more than 24 hr to avoid repeated freeze thawing.
Using a commercial DNA/RNA co-extraction kit, 200. mu.L of the sample to be tested was taken and the sample was treated according to the protocol. The positive control and the negative control were not involved in the extraction and were used directly as templates.
(2) Reagent preparation (performed in reagent preparation area):
taking out the components in the kit, standing at room temperature, and uniformly mixing the components for later use after the temperature of the components is balanced to the room temperature;
calculating the number of required reagents N (the number of the reagents (N) ═ number of the samples to be detected (N) + Positive Control (PC) + Negative Control (NC)) according to the number of the samples to be detected, and preparing a reaction mixed solution according to the following table 4:
TABLE 4 reaction mixture
Reaction mixture components The dosage of each part of the medicine is
CVD main reaction liquid 5μL
CVD reaction liquid 2μL
RNase-Free H2O 3μL
Total volume 10μL
And (3) fully and uniformly mixing the reaction mixed solution, and subpackaging the mixture into N PCR reaction tubes according to 10 mul/tube.
The prepared reagent is transferred to a sample processing area for use.
(3) Establishing a fluorescent quantitative PCR system containing the primer probe composition by taking the nucleic acid extracting solution as a template: and adding 10 mu L of the sample to be detected into a PCR amplification tube filled with 10 mu L of prepared PCR-mixed solution according to a certain sequence, respectively taking the extracted sample nucleic acid solution to be detected, positive control and negative control, detecting fluorescent quantitative PCR on a fluorescent PCR instrument, detecting the PCR reaction mixed solution containing the template through a fluorescent quantitative PCR detection system, and judging whether the patient is infected with the novel coronavirus according to the obtained different fluorescent signal data.
(4) PCR amplification (in the amplification and analysis region)
And (3) placing the PCR reaction tube into a sample groove of the amplification instrument, and setting the sample name.
Fluorescence detection channel selection: and selecting three channels of FAM, VIC and CY5 for real-time fluorescent signal acquisition. Cycle parameter settings refer to table 5:
TABLE 5 circulation parameters
Figure BDA0002554607850000081
Note: finally, fluorescence signals were collected at 58 ℃ for 60 seconds of extension.
And after the setting is finished, saving the file and operating the reaction program.
(5) Analysis of results (set up with reference to the instructions for use of the respective apparatus, the following analyses are exemplified for the ABI series apparatus):
and automatically storing results after the reaction is finished, and analyzing the amplification curves of the detection target and the internal standard respectively. Adjusting the Start value, the End value and the Threshold value of Baseline according to the analyzed image (the user can adjust the Start value to be 3-15 and the End value to be 5-20 according to the actual situation, adjusting the amplification curve of negative control to be straight or lower than a Threshold line), clicking Analyze to Analyze, enabling each parameter to meet the requirements in the quality control of the table 6, and then recording the qualitative result under a Plate window.
TABLE 6 quality control
Figure BDA0002554607850000091
Note: the requirements are required to be met in the same experiment, otherwise, the experiment is invalid, and the detection is required to be carried out again;
(6) and (4) interpretation of results:
referring to Table 7, the results were analyzed in the case where the apparatus was normal, and the negative and positive controls were normal (FAM channel represents COVID-19ORF1ab gene, VIC channel represents COVID-19N gene, CY5 channel represents internal standard):
TABLE 7 results interpretation criteria
Figure BDA0002554607850000092
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
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Claims (10)

1. A novel primer probe composition for coronavirus nucleic acid detection, comprising:
the first primer probe group comprises a forward primer ORF1ab-F, a probe ORF1ab-P and a reverse primer ORF1ab-R, and the nucleotide sequences of the first primer probe group are respectively shown as SEQ ID Nos. 1-3;
a second primer probe group, which comprises a forward primer N-F, a probe N-P and a reverse primer N-R, wherein the nucleotide sequences of the second primer probe group are respectively shown as SEQID Nos. 4-6;
the third primer probe set comprises a forward primer Rpp30-F, a probe Rpp30-P and a reverse primer Rpp30-R, and the nucleotide sequences of the third primer probe set are respectively shown as SEQ ID Nos. 7-9.
2. The primer probe composition for detecting coronavirus nucleic acid as claimed in claim 1, wherein the 5 'end of the probe ORF1ab-P, the 5' end of the probe N-P and the 5 'end of the probe Rpp30-P are labeled with a fluorescent group, and the 3' end of the probe is labeled with a fluorescence quenching group.
3. The primer-probe composition for detecting coronavirus nucleic acid as claimed in claim 2, wherein the fluorescent group is at least 1 selected from FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red and CY 3.
4. The primer-probe composition for detecting coronavirus nucleic acid of claim 2, wherein the fluorescence quenching group is at least 1 selected from the group consisting of MGB, BHQ-1, BHQ-2, BHQ-3 and TAMRA.
5. A novel coronavirus nucleic acid detection kit comprising the primer probe composition as defined in any one of claims 1 to 4, which comprises a primer probe composition container, an internal and external quality control container, a reverse transcription reaction and PCR amplification reaction container, wherein the primer probe composition container contains a primer probe dry powder or solution having a nucleotide sequence as shown in SEQ ID Nos. 1 to 9; the inner and outer quality control containers contain dry powder or solution with nucleotide sequences shown as SEQ ID No. 10-12; the reverse transcription reaction and PCR amplification reaction vessel comprises reverse transcriptase, DNA polymerase, auxiliary reinforcing agent and buffer solution.
6. The kit for detecting coronavirus nucleic acid according to claim 5, wherein the pH of the buffer solution is controlled to 7.5-9.5.
7. The novel coronavirus nucleic acid detection kit according to claim 5, further comprising a positive control group and a negative control group, wherein the positive control group is a novel coronavirus COVID-19 nucleic acid or a pseudovirion; the negative control group is human source sample housekeeping gene pseudovirion.
8. A method for the detection of novel coronavirus nucleic acids using a primer probe composition as defined in any one of claims 1 to 4 for non-diagnostic purposes, comprising the steps of:
(1) extracting and purifying nucleic acid of the novel coronavirus sample to obtain a nucleic acid extracting solution;
(2) establishing a fluorescent quantitative PCR system containing the primer probe composition by taking the nucleic acid extracting solution as a template;
(3) and detecting the PCR reaction mixed solution containing the template by a fluorescent quantitative PCR detection system, and judging whether the patient is infected with the novel coronavirus according to the obtained different fluorescent signal data.
9. The method for detecting the nucleic acid of the novel coronavirus according to claim 8, wherein the fluorescent quantitative PCR system comprises a primer-probe mixture of the novel coronavirus ORF1ab, the N gene and the human-derived sample housekeeping gene in step (2).
10. The method for detecting novel coronavirus nucleic acid according to claim 8, wherein the human-derived sample housekeeping gene is selected from at least 1 of protein subunits Rpp 20, Rpp30, Rpp40, GAPDH, and β -actin of Rnase P.
CN202010588373.0A 2020-06-24 2020-06-24 Novel primer probe composition, kit and method for coronavirus nucleic acid detection Pending CN111621604A (en)

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