CN112626272A - Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit - Google Patents
Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit Download PDFInfo
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Abstract
The invention provides a novel coronavirus SARS-CoV-2 detection and molecular typing method and a kit. The method combines one-step method of multiple RT-PCR and HRM analysis, and realizes the detection and typing of SARS-CoV-2. The detection method comprises the following steps: 1) extracting nucleic acid of a sample; 2) completing one-step multiple RT-PCR reaction in a special primer group and a reagent; 3) HRM analysis interprets SARS-CoV-2 detection results and molecular typing results. The kit provided by the invention comprises special primers for multiplex detection and molecular typing of SARS-CoV-2, and also comprises reaction components Master Mix (RNA reverse transcriptase, polymerase, amplification buffer, dNTP and EvaGreen fluorescent dye), positive control and negative control.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, relates to a pathogen detection method, and particularly relates to a novel coronavirus SARS-CoV-2 detection and molecular typing method and a kit.
Background
Coronaviruses (CoVs), which belong to the family of coronaviridae, are a large family of viruses that are widely found in nature and that infect humans and animals, and are named when the virion surface is observed under an electron microscope to have coronaries-like fibers. The genome of the coronavirus is large, the single-stranded positive-strand gene sequence of the coronavirus is 26-32 kb in length, and the coronavirus is an RNA virus with the longest natural sequence.
To date, the coronaviridae only infect vertebrates, and mostly cause respiratory tract and digestive tract symptoms of human and animals, and other organ and nervous system symptoms are also occasionally reported. Human coronavirus infection mainly comprises upper respiratory tract, lower respiratory tract and gastrointestinal tract symptoms, mild symptoms such as common cold, cough, fever, diarrhea and the like, severe symptoms such as bronchitis and pneumonia are developed, and symptoms such as renal failure, acute respiratory distress syndrome and the like are accompanied.
Persistent epidemic new Coronavirus pneumonia (Coronavir disease 2019, COVID-19) caused by Severe acute respiratory syndrome Coronavirus type 2 (SARS-CoV-2) now poses an unprecedented threat to global public health and economy. SARS-CoV-2 infection mainly causes respiratory infectious diseases mainly including pulmonary diseases, and also causes serious complications such as acute myocardial injury and acute respiratory distress syndrome, even death. Although the mortality rate of Severe acute respiratory syndrome coronavirus (SARS-CoV) infection appeared in 2003 and Middle east respiratory syndrome coronavirus (MERS-CoV) infection appeared in 2012 was higher than that of SARS-CoV-2, the spread rate of SARS-CoV-2 was much higher than that of MERS-CoV and SARS-CoV. In addition, no specific therapeutic drug for SARS-CoV-2 exists, and a prophylactic vaccine is not widely used. In the case of global outbreaks, rapid detection of SARS-CoV-2 infection as early as possible (preferably at the earliest stage of symptoms) is critical in controlling viral spread, and in particular, the clinical manifestations of SARS-CoV-2 infected patients are very similar to those of other seasonal respiratory tract infections such as influenza and are difficult to distinguish.
Therefore, there is an urgent need for a specific and sensitive method for detecting, identifying and screening SARS-CoV-2 in order to determine COVID-19 positive cases at an early stage before clinical symptoms appear in the patient. Currently, SARS-CoV-2 detection is mainly based on two diagnostic strategies: firstly, detecting virus RNA in respiratory tract specimens (nasopharyngeal swab, pharyngeal swab, saliva, sputum and lower respiratory tract secretion) by a molecular diagnosis method; the second is a serological or antibody test that uses a blood sample to detect specific antibodies. Serological tests allow the patient to be monitored for immune response and antibody production during the acute phase (first week of onset) and the convalescent phase (2-4 weeks after onset). Serotype detection is therefore particularly useful for retrospective studies of COVID-19 infection, but not for the identification of early stage SARS-CoV-2 infection. Since SARS-CoV-2 genome sequence information is published for the first time, a series of Nucleic acid amplification methods (NAAT) are rapidly established by virtue of the advantages of short time consumption, simple and convenient operation, automation and the like for detecting SARS-CoV-2 virus RNA, and comprise Next Generation Sequencing (NGS), real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) and a method based on a regular interval short palindromic repeat (CRISPR) mechanism. In the early stages of a new outbreak of a coronary epidemic, the NGS technology recognizes and discovers the pathogen SARS-CoV-2 in time, without the knowledge of the causative pathogen. In addition to being used to confirm the suspected COVID-19 disease, NGS technology can also help us to monitor mutations that may occur in the SARS-CoV-2 genome, which is important for understanding the pathogenic mechanism and evolution of the SARS-CoV-2 virus. Although the price of NGS is decreasing year by year, the cost is still high, the whole operation procedure is complicated (including time-consuming and complex library preparation and data processing), professional personnel are required for data analysis, and the disposable sample is limited, which greatly limits the application of NGS in large-scale outbreak. The real-time RT-PCR detection of SARS-CoV-2 is a detection method with strong specificity and high sensitivity, is recommended by the world health organization as an effective and simple method, and is also a gold standard method for identifying COVID-19 pandemic in current research and clinical laboratories. Although real-time RT-PCR assays have become widely used, the increasing demand for assays on a global scale has led to a shortage of reagents and associated consumables, thereby affecting the diagnosis of more and more suspected COVID-19 infections. To meet the unprecedented demand for SARS-CoV-2 diagnosis worldwide, many alternative methods, such as LAMP, RPA and CRISPR, have been developed to achieve equivalent levels of detection of viral RNA. Although these detection methods are faster, simpler, less costly and use less time to confirm the diagnosis of COVID-19 than NGS, they do not provide information on the subtype and mutation of SARS-CoV-2. Therefore, there is an urgent need to develop and establish a comprehensive diagnostic method, which can be used not only for virus identification, but also for typing and mutation detection of SARS-CoV-2 genome, and provide information for epidemic prevention and control in time.
High Resolution Melting curve analysis (HRM) is a new molecular diagnostic technique for detecting gene mutation and genotyping by combining with saturated fluorescent dye, unlabeled probe and real-time fluorescent quantitative PCR, which has emerged in recent years. HRM is based on real-time fluorescence quantitative PCR, saturated fluorescent dye is added into the system, a high-precision instrument is utilized, the DNA melting process is monitored in real time through high-resolution melting of PCR products, and the tiny difference of DNA sequences is analyzed according to the characteristic change of a melting curve. Because HRM has the advantages of rapidness, accuracy, high throughput, strong specificity, high sensitivity, low cost, realization of real closed-tube operation and the like, the HRM is widely applied to the fields of sequence analysis, genotyping, mutation site scanning, single nucleotide polymorphism analysis, clinical detection and the like.
Disclosure of Invention
The invention aims to provide a special primer with strong specificity and high sensitivity for SARS-CoV-2 detection and molecular typing methods.
The special primer for SARS-CoV-2 detection and molecular typing selects interspecific specific and intraspecies conserved gene or region as SARS-CoV-2 virus detection target gene (including ORFla gene, N gene and E gene), selects related mutation site capable of accurately classifying SARS-CoV-2 into S, L, V, G, GH and GR types (including T8782G, C28144T,G11083T, G26144T, C241T, C3037T, A23043G, G28882A, and G25563T) region (region: (region A), (region B), (region C), (regionhttps://www.gisaid.org/references/statementss-clarifications/clade-and- lineage-nomenclature-aids-in-genomic-epidemiology-of-active-hcov-19-viruses/) The PCR product is used as a detection target for molecular typing of SARS-CoV-2 virus, and simultaneously, a human RNase P gene is selected as an internal control for monitoring the extraction condition of sample nucleic acid. First from GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) And GISAID database: (https://www.gisaid.org) The SARS-CoV-2 is downloaded and used as a representative strain gene sequence of a reference sequence after complete annotation, the reference sequence and an nr database of NCBI are subjected to nucleic acid sequence BLAST (https:// BLAST. Specific amplification primers were designed on both sides of the detection target using SnapGene software and the specificity of the primers was verified using the NCBI's on-line primer tool (https:// www.ncbi.nlm.nih.gov/tools/primer blast /). The Melting temperature (Tm) of the amplified product was predicted using oligocalc (http:// biolols. nucic. norwestern. edu/oligocalc. html) and UMELT online software (https:// www.dna.utah.edu/UMELT. html). 6 groups of primer pairs are designed aiming at each detection site, and the optimal primer pair capable of accurately distinguishing the wild type and the mutant type is screened out. In addition, non-specific GC tails are added to some primers, so that Tm values among amplification products do not overlap, and different melting curves can be detected and analyzed simultaneously. And finally, selecting an optimal primer pair capable of accurately distinguishing the wild type and the mutant type to form a final multiple HRM analysis system.
The special primer for detecting SARS-CoV-2 and molecular typing may be one or several of 13 primer sets, and the 13 primer sets are shown in SEQ ID No.1-SEQ ID No. 26.
Wherein, the special primer for detecting SARS-CoV-2 is shown in SEQ ID NO.1-SEQ ID NO. 8.
Wherein, the special primer for detecting the molecular typing of SARS-CoV-2 is shown as SEQ ID NO.9-SEQ ID NO. 26.
Among them, the molecular typing for detecting SARS-CoV-2 means that SARS-CoV-2 can be accurately classified into S, L, V, G, GH type and GR type.
The special primer for SARS-CoV-2 detection and molecular typing method is characterized by that the described primer is a characteristic primer group with 13 target spots, one primer group is formed from two primers, one is forward primer and another is reverse primer. The total number of 13 primer sets is 13, and multiple detection reactions are respectively realized aiming at 13 detection targets, wherein SEQ ID NO.1-SEQ ID NO.8 is used for detecting SARS-CoV-2, SEQ ID NO.9-SEQ ID NO.26 is used for molecular typing of SARS-CoV-2, and the sequence information of the primers is shown in Table 1.
The 13 specific detection targets of the invention comprise: taking interspecific specific and intraspecies conserved genes or regions (comprising ORF1a gene, N gene and E gene) as target genes for SARS-CoV-2 virus detection; 9 different targeting regions cover 9 important mutation sites such as T8782G, C28144T, G11083T, G26144T, C241T, C3037T, A23043G, G28882A and G25563T and are used for molecular typing of SARS-CoV-2, so that 6 types of SARS-CoV-2 virus S, L, V, G, GH and GR are accurately distinguished; the human RNase P gene is included in the detection system as an internal control for monitoring the extraction condition of the sample nucleic acid.
Another object of the present invention is to provide a method for screening primers specific for detecting SARS-CoV-2 and molecular typing, comprising the steps of:
1) and (3) selecting a detection target: selecting interspecific specific and intraspecies conserved genes or regions as target genes (including ORF1a gene, N gene and E gene) for SARS-CoV-2 virus detection; selecting the region of related mutation sites (including T8782G, C28144T, G11083T, G26144T, C241T, C3037T, A23043G, G28882A and G25563T) which can accurately classify SARS-CoV-2 into S, L, V, G, GH and GR types as the detection target of SARS-CoV-2 virus molecular classification; meanwhile, selecting a human RNase P gene as an internal control for monitoring the extraction condition of the sample nucleic acid;
2) designing a primer: first from GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) And GISAID database: (https://www.gisaid.org) The gene sequence of a representative strain in which SARS-CoV-2 was completely annotated as a reference sequence will beAnd (3) carrying out nucleic acid sequence BLAST (https:// blast.ncbi.nlm.nih.gov/blast.cgi) on the reference sequence and an nr database of NCBI, downloading and comparing the obtained result, and obtaining more detection target gene sequences. Specific amplification primers were designed on both sides of the detection target using SnapGene software and the specificity of the primers was verified using the NCBI's on-line primer tool (https:// www.ncbi.nlm.nih.gov/tools/primer blast /). Predicting the Melting temperature (Tm) of the amplified product using oligocalc (http:// biolols. nucic. norwestern. edu/oligocalc. html) and UMELT online software (https:// www.dna.utah.edu/UMELT. html);
3) primer screening: 6 groups of primer pairs are designed aiming at each detection site, and the optimal primer pair capable of accurately distinguishing the wild type and the mutant type is screened out. In addition, nonspecific GC tails are added into some primers, so that Tm values among amplification products are not overlapped, and different melting curves can be detected and analyzed simultaneously;
4) determination of multiplex detection primer set: and selecting an optimal primer pair capable of accurately distinguishing the wild type and the mutant type to form a final 4-well 13-weight HRM analysis system.
TABLE 1 primer sequence information
The addition of additional G or Gc bases 5' to the a primer is underlined.
b the amount of primer contained in the multiple reaction wells.
The second purpose of the present invention is to provide a method for detecting SARS-CoV-2 and molecular typing.
The assay of the invention is used for non-diagnostic disease purposes.
The SARS-CoV-2 detecting and molecular typing method includes the following steps:
(1) completing the extraction of the test sample nucleotide by using a commercial viral RNA extraction kit;
(2) taking the nucleotide of a sample to be detected as a template, preparing a one-step HRM amplification reaction system under the guidance of the special primer group, and performing specific amplification of each detection reaction hole;
3) in the PCR process, due to the sequence specificity of a target sequence, the base content difference of different PCR products can be caused, PCR amplicons are heated under the action of a saturated dye according to the property of a gene sequence, data integration and image drawing are carried out on a detection result by monitoring the change of fluorescence intensity in the heating process in real time, a melting curve of the PCR products is generated, the difference of the gene sequence in the PCR products is judged according to the difference of the melting curve, and the SARS-CoV-2 virus is determined and accurately typed.
The third purpose of the present invention is to provide a kit for detecting SARS-CoV-2 and molecular typing.
The kit for detecting SARS-CoV-2 and molecular typing provided by the invention comprises special primers for multiplex SARS-CoV-2 detection and molecular typing.
The kit for detecting SARS-CoV-2 and molecular typing provided by the invention comprises one or more groups of special primer groups shown in SEQ ID NO.1-SEQ ID NO. 26.
The kit for detecting SARS-CoV-2 provided by the invention comprises the special primers shown in SEQ ID NO.1-SEQ ID NO. 8.
The kit for detecting the molecular typing of SARS-CoV-2 provided by the invention comprises SEQ ID NO.9-SEQ ID NO. 26.
The kit also comprises reaction components Master Mix (RNA reverse transcriptase, polymerase, amplification buffer, dNTP and EvaGreen fluorescent dye), a positive control and a negative control, wherein the positive control is a wild type or mutant type positive sample of each detection target spot, and the negative control is ddH2O。
The sample of the novel coronavirus is derived from a human nasopharynx swab, saliva, a sputum sample, an alveolar lavage fluid sample, a blood sample, a lower respiratory secretion, a stool sample or an environmental sample.
Wherein the final concentration of the primer in the PCR reaction system is shown in Table 1.
The fourth purpose of the invention is to provide the application of the kit in detecting SARS-CoV-2 and molecular typing.
The application of the invention comprises the following steps:
1) patient respiratory specimens (nasopharyngeal swabs, pharyngeal swabs, saliva, sputum, and lower respiratory secretions) were collected using a dedicated viral collection tube.
2) Viral RNA was extracted from the samples according to the QIAamp Viral RNA Mini kit instructions, while RNase inhibitor was added to the RNA storage tubes. (the above steps may be performed using other nucleic acid extraction kits or methods to accomplish viral RNA extraction).
3) SARS-CoV-2 detection (Assay1) and molecular typing (Assay2-4) were carried out in 4 Assay reaction wells using the sample viral RNA obtained in the above procedure as a template. Wherein the 20 mu L one-step multiple reaction system comprises: 10 μ L of Reaction Mix, 1uL of 20 × Eventreen, 1 μ L
III RT/
Optimal amplification concentration of each primer in Taq Mix, Assay (Table 1), sample viral RNA template 2. mu.L, ddH2O to 20. mu.L. Positive control and negative control reaction tubes were introduced simultaneously for each detection reaction.
4) The one-step multiplex amplification reaction and HRM analysis are carried out in a QuantStaudio 6Flex Real-Time PCR System. The conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; after a subsequent 2 min incubation at 95 ℃, denaturation at 94 ℃ for 30 sec, annealing at 53 ℃ for 15 sec, and extension at 68 ℃ for 15 sec, 30 cycles of amplification were performed, followed by incubation at 60 ℃ for 1 min, followed by slow temperature increase at a rate of 0.025 ℃/s to 95 ℃ and continuous collection of fluorescence signals.
5) After the reaction is finished, the QuantStaudio 6and 7Flex Real-Time PCR software v1.0 is used for analysis, and the software can automatically generate a melting curve and a Tm value corresponding to the amplicon. The results were judged by comparison with wild type or mutant positive controls. If the drug resistance site of the sample to be detected is the same as that of the wild type control sample, the shape of the melting curve is unchanged, and if the sample to be detected is different from that of the wild type control sample, a mutation can be formed, and the shape of the melting curve can be correspondingly changed.
6) And (4) interpretation of results: judging the detection result, if the sample is positive, three specific product peaks are generated by three targets of ORF1a gene, N gene and E gene in the detection hole Assay1, and the Tm values of the three product peaks are consistent with that of a positive control (FIG. 2A); if only 1-2 of the three detection targets generate product peaks consistent with the positive control, the sample is judged to have uncertain results and the experiment needs to be repeated again; otherwise, the sample is judged to be SARS-CoV-2 negative. And (3) judging the typing result, comparing the detection product peaks of the 9 targets in the Assay2-4 with a positive control, determining the mutation condition of each mutation site (shown in figures 2B-D), and finally integrating the site mutation conditions of the 9 targets to determine the molecular typing of SARS-CoV-2.
The method of the present invention can detect SARS-CoV-2 virus from clinical sample fast, specifically and sensitively and perform molecular typing. Compared with other existing SARS-CoV-2 detecting technology and the similar technology, the technical scheme of the invention has the following advantages: firstly, the method improves and optimizes HRM reaction conditions for the first time, and breakthroughs the use of a one-step method multiple PCR technology and HRM analysis for combination, so that the direct detection and typing after nucleic acid extraction from clinical samples are realized, RNA reverse transcription is not required to be additionally carried out, the detection time is greatly shortened, and the consumption of reagents is reduced; compared with the traditional PCR or other molecular detection technologies, the HRM technology is a high-throughput gene screening technology for analyzing PCR products by monitoring the change of a melting curve in real time, is not limited by the mutation position and the mutation type of a detection target, does not need to synthesize expensive sequence-specific probes, greatly reduces the detection cost, can quickly and sensitively analyze an experimental result by directly passing through a high-resolution melting curve after the reaction is finished, obtains the mutation information of a detection target site and directly obtains a typing result; secondly, saturated dye EvaGreen is adopted in the HRM analysis experiment, and the saturated dye EvaGreen can not inhibit PCR reaction, can be directly added into a PCR reaction system before the reaction starts to participate in the PCR process, and can be directly used for HRM analysis without transferring to other analysis devices or opening the cover to add dye after the reaction is finished, so that closed-tube operation is really realized, the pollution possibly caused by opening the cover is avoided, false positive results are caused, and the accuracy and the reliability of the experiment results are improved; finally, the heating and cooling in the analysis process can not cause destructive damage to the DNA structure, the subsequent cooling can lead the DNA to be renatured, the renatured DNA can be directly used for subsequent research (such as sequencing verification results), the time, the manpower and the material resources are greatly saved, and unnecessary waste is avoided.
For the terms appearing herein, further explanation and explanation are provided:
HRM: high resolution melting curve
And (3) PCR: polymerase chain reaction
Amplifying buffer: one-step amplification reaction buffer solution
dNTP: oligonucleotide for one-step amplification reaction
EvaGreen fluorescent dye: saturated fluorescent dyes for HRM analysis
Reaction Mix: reaction premix
Assay: reaction well
QIAamp Viral RNA Mini kit: viral RNA extraction kit
Drawings
FIG. 1, the principle and the procedure of SARS-CoV-2 detection and molecular typing
FIG. 2, schematic representation of the results of SARS-CoV-2 detection and molecular typing
FIG. 2A: assay1 test results
FIG. 2B: assay2 test results
FIG. 2C: assay3 test results
FIG. 2D: assay4 test results
Detailed Description
Example 1 is carried out on the premise of the invention, and a detailed implementation mode and a specific operation process are given. The specific embodiments and operations described herein are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. The following is a description of the embodiments and specific procedures of the examples for SARS-CoV-2 detection and molecular typing in clinical use in hospitals.
1) Patient respiratory specimens (nasopharyngeal swabs, pharyngeal swabs, saliva, sputum, and lower respiratory secretions) were collected using a dedicated viral collection tube.
2) Viral RNA was extracted from the samples according to the QIAamp Viral RNA Mini kit instructions, while RNase inhibitor was added to the RNA storage tubes. (the above steps can be performed by other nucleic acid extraction kits or methods)
3) SARS-CoV-2 detection (Assay) and molecular typing (Assay2-4) were carried out in 4 Assay reaction wells using the sample viral RNA obtained in the above procedure as a template. Wherein the 20 mu L one-step multiple reaction system comprises: 10 μ L of Reaction Mix, 1 μ L of 20 × Eventreen, 1 μ L
III RT/
Optimal amplification concentration of each primer in Taq Mix, Assay (Table 1), 2. mu.L of sample viral RNA template, ddH2Make up to 20. mu.L of O. Positive control and negative control reaction tubes were introduced simultaneously for each detection reaction.
4) The one-step multiplex amplification reaction and HRM analysis are carried out in a QuantStaudio 6Flex Real-Time PCR System. The conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; after a subsequent 2 min incubation at 95 ℃, denaturation at 94 ℃ for 30 sec, annealing at 53 ℃ for 15 sec, and extension at 68 ℃ for 15 sec, 30 cycles of amplification were performed, followed by incubation at 60 ℃ for 1 min, followed by slow temperature increase at a rate of 0.025 ℃/s to 95 ℃ and continuous collection of fluorescence signals.
5) After the reaction is finished, the QuantStaudio 6and 7Flex Real-Time PCR software v1.0 is used for analysis, and the software can automatically generate a melting curve and a Tm value corresponding to the amplicon. The results were judged by comparison with wild type or mutant positive controls. If the drug resistance site of the sample to be tested is the same as that of the wild type control sample, the shape of the melting curve is unchanged, and if the sample to be tested is different from that of the wild type control sample, a mutation is formed, and the shape of the melting curve is also changed correspondingly (fig. 2).
6) And (4) interpretation of results: judging the detection result, if the sample is positive, three specific product peaks are generated by three targets of ORF1a gene, N gene and E gene in the detection hole Assay1, and the Tm values of the three product peaks are consistent with that of a positive control (FIG. 2A); if only 1-2 of the three detection targets generate product peaks consistent with the positive control, the sample is judged to have uncertain results and the experiment needs to be repeated again; otherwise, the sample is judged to be SARS-CoV-2 negative. And (3) judging the typing result, comparing the detection product peaks of the 9 targets in the Assay2-4 with a positive control, determining the mutation condition of each mutation site (shown in figures 2B-D), and finally integrating the site mutation conditions of the 9 targets to determine the molecular typing of SARS-CoV-2.
Example 2 determination of the Performance of the kit
1. Accuracy verification
The results of selecting RNA extracted from 5 SARS-CoV-2 isolates infected with cells and 6 clinical specimens of positive patients and testing according to the method and procedure of example 1 above show that the Assay1 of these samples can accurately identify three SARS-CoV-2 target genes and determine that these samples are positive for SARS-CoV-2; these samples were then typed into 5 different GISAID classes, including L, S, V, G and GR, based on the results of the 9-labeled mutation sites of Assay 2-4. And simultaneously sequencing the 9 marked mutation sites of the samples by using a PCR-sequencing method, wherein the result shows that the typing result of the method is consistent with the typing result obtained by the PCR-sequencing method. In conclusion, the method has good detection and typing performance.
2. Specificity verification
The specificity of the method was assessed by selecting pathogens common to respiratory infections, including adenovirus, enterovirus, human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1, MERS-CoV, bocavirus, metapneumovirus types A and B, rhinovirus, influenza A H1N1, influenza A H3N2, influenza B, parainfluenza viruses (types 1 to 4), respiratory syncytial viruses (types A and B), Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter mansonii, Escherichia coli, Streptococcus pneumoniae and Neisseria meningitidis, and testing was performed with reference to the method and procedures of example 1 above, and the results showed that Assay1-4 of the method was non-cross-reactive to all pathogens tested, the method is proved to have good specificity.
3. Sensitivity detection
The sensitivity was assessed using a dilution-by-fold gradient positive plasmid, tested according to the method and procedure described above in example 1, with 11 replicates per dilution, and the final detection limit determined using probit regression analysis at 95% detection level. The results show that the detection limit is below 10 copies/reaction for all targets detected by this method (table 2).
TABLE 2 detection limits for the respective detection targets
aCI, confidence interval.
Sequence listing
<110> institute of pathogenic biology of Chinese academy of medical sciences
<120> a novel coronavirus SARS-CoV-2 detection and molecular typing method and kit
<141> 2020-12-25
<160> 26
<170> SIPOSequenceListing 1.0
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<211> 33
<212> RNA
<213> ORF1ab
<400> 1
<210> 2
<211> 33
<212> RNA
<213> ORF1ab
<400> 2
<210> 3
<211> 33
<212> RNA
<213> N
<400> 3
<210> 4
<211> 33
<212> RNA
<213> N
<400> 4
<210> 5
<211> 33
<212> RNA
<213> E
<400> 5
<210> 6
<211> 33
<212> RNA
<213> E
<400> 6
<210> 7
<211> 33
<212> RNA
<213> RNase P
<400> 7
<210> 8
<211> 33
<212> RNA
<213> RNase P
<400> 8
<210> 9
<211> 33
<212> RNA
<213> ORF8-28144
<400> 9
<210> 10
<211> 33
<212> RNA
<213> ORF8-28144
<400> 10
<210> 11
<211> 33
<212> RNA
<213> S-23403
<400> 11
<210> 12
<211> 33
<212> RNA
<213> S-23403
<400> 12
<210> 13
<211> 33
<212> RNA
<213> ORF3a-26144
<400> 13
<210> 14
<211> 33
<212> RNA
<213> ORF3a-26144
<400> 14
<210> 15
<211> 33
<212> RNA
<213> ORF1ab-3037
<400> 15
<210> 16
<211> 33
<212> RNA
<213> ORF1ab-3037
<400> 16
<210> 17
<211> 33
<212> RNA
<213> ORF3a-25563
<400> 17
<210> 18
<211> 33
<212> RNA
<213> ORF3a-25563
<400> 18
<210> 19
<211> 33
<212> RNA
<213> 5'UTR-241
<400> 19
<210> 20
<211> 33
<212> RNA
<213> 5'UTR-241
<400> 20
<210> 21
<211> 33
<212> RNA
<213> ORF1ab-11083
<400> 21
<210> 22
<211> 33
<212> RNA
<213> ORF1ab-11083
<400> 22
<210> 23
<211> 33
<212> RNA
<213> ORF1ab-8782
<400> 23
<210> 24
<211> 33
<212> RNA
<213> ORF1ab-8782
<400> 24
<210> 25
<211> 33
<212> RNA
<213> N-28882
<400> 25
<210> 26
<211> 33
<212> RNA
<213> N-28882
<400> 26
Claims (9)
1. The special primer for detecting SARS-CoV-2 and molecular typing is one or several of 13 primer sets, and the 13 primer sets are shown in SEQ ID No.1-SEQ ID No. 26.
2. The special primer for detecting SARS-CoV-2 according to claim 1, which is represented by SEQ ID NO.1-SEQ ID NO. 8.
3. The special primer for detecting the molecular typing of SARS-CoV-2 as claimed in claim 1, which is represented by SEQ ID NO.9-SEQ ID NO. 26.
4. The primer set of claim 1, wherein the molecular typing of SARS-CoV-2 is carried out by differentiating SARS-CoV-2 into S, L, V, G, GH types and GR types.
5. The method for screening the special primer according to claim 1, comprising the following steps:
1) and (3) selecting a detection target: selecting interspecific specific and intraspecies conserved gene or region as a target gene for detecting SARS-CoV-2 virus; selecting a relative mutation site region which can accurately divide SARS-CoV-2 into S, L, V, G, GH types and GR types as a detection target of SARS-CoV-2 virus molecular typing; meanwhile, selecting a human RNase P gene as an internal control for monitoring the extraction condition of the sample nucleic acid;
2) designing a primer: firstly, downloading SARS-CoV-2 from a GenBank database and a GISAID database, taking complete annotation as a representative strain gene sequence of a reference sequence, carrying out nucleic acid sequence BLAST on the reference sequence and an nr database of NCBI, downloading and comparing the obtained result to obtain more detection target gene sequences, designing specific amplification primers on two sides of a detection target by using SnapGene software, verifying the specificity of the primers by using an online primer tool of the NCBI, and predicting the melting temperature of an amplification product by using oligocalc and UMELT online software;
3) primer screening: 6 groups of primer pairs are designed aiming at each detection site, and the optimal primer pair capable of accurately distinguishing the wild type and the mutant type is screened out,
4) determination of multiplex detection primer set: and selecting an optimal primer pair capable of accurately distinguishing the wild type and the mutant type to form a final 4-well 13-weight HRM analysis system.
6. A method for detecting SARS-CoV-2 and molecular typing for the purpose of non-diagnosing disease, comprising the steps of:
(1) completing the extraction of the test sample nucleotide by using a commercial viral RNA extraction kit;
(2) taking the nucleotide of a sample to be detected as a template, preparing a one-step HRM amplification reaction system under the guidance of the special primer group, and performing specific amplification of each detection reaction hole;
3) in the PCR process, due to the sequence specificity of a target sequence, the base content difference of different PCR products can be caused, PCR amplicons are heated under the action of a saturated dye according to the property of a gene sequence, data integration and image drawing are carried out on a detection result by monitoring the change of fluorescence intensity in the heating process in real time, a melting curve of the PCR products is generated, the difference of the gene sequence in the PCR products is judged according to the difference of the melting curve, and the SARS-CoV-2 virus is determined and accurately typed.
7. A kit for detecting SARS-CoV-2 and molecular typing comprises one or more groups of special primer groups shown in SEQ ID NO.1-SEQ ID NO. 26.
8. The kit according to claim 7, further comprising reaction components Master Mix (RNA reverse transcriptase, polymerase, amplification buffer, dNTP and EvaGreen fluorescent dye), a positive control and a negative control, wherein the positive control is a wild-type or mutant-type positive sample of each detection target, and the negative control is ddH2O。
9. Use of the kit of claim 7 for detecting SARS-CoV-2 and molecular typing comprising the steps of:
1) using a special virus collecting tube to collect the respiratory tract specimen of a patient,
2) extracting Viral RNA from the sample according to the QIAamp Viral RNA Mini kit operating instructions, simultaneously adding RNase inhibitor into the RNA preservation tube,
3) using the virus RNA as template to realize SARS-CoV-2 detection and molecular typing in 4 Assay reaction holes,
wherein the 20 mu L one-step multiple reaction system comprises: 10 μ L of Reaction Mix, 1 μ L of 20 × Eventreen, 1 μ L
III RT/
Optimal amplification concentration of each primer in Taq Mix and Assay, 2 mu L of sample virus RNA template and 2 mu L of ddH2O are complemented to 20 mu L, a positive control reaction tube and a negative control reaction tube are introduced into each detection reaction simultaneously,
4) performing one-step multiplex amplification reaction and HRM analysis on a QuantStaudio 6Flex Real-Time PCR System, wherein the conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; followed by incubation at 95 ℃ for 2 min, denaturation at 94 ℃ for 30 sec, annealing at 53 ℃ for 15 sec, extension at 68 ℃ for 15 sec, incubation at 60 ℃ for 1 min after 30 cycles of amplification, followed by slow temperature increase at 0.025 ℃ per second to 95 ℃ and continuous collection of fluorescent signal,
5) after the reaction is finished, the QuantStaudio 6and 7Flex Real-Time PCR software v1.0 is used for analysis, software can automatically generate a melting curve and a Tm value corresponding to an amplicon, the result is judged by comparing the melting curve and the Tm value with a wild type or mutant type positive control, if the drug-resistant site of the sample to be detected is the same as the wild type control sample, the shape of the melting curve is unchanged, if the sample to be detected is different from the wild type control sample, a mutation can be formed, the shape of the melting curve can be correspondingly changed,
6) and (4) interpretation of results: judging the detection result, if the sample is positive, generating three specific product peaks by three targets of ORF1a gene, N gene and E gene in a detection hole Assay1, wherein the Tm values of the three product peaks are consistent with that of a positive control; if only 1-2 of the three detection targets generate product peaks consistent with the positive control, the sample is judged to have uncertain results and the experiment needs to be repeated again; otherwise, the sample is judged to be SARS-CoV-2 negative. And (3) judging the typing result, comparing the detection product peaks of the 9 targets in the Assay2-4 with a positive control, determining the mutation condition of each mutation site, and finally integrating the site mutation conditions of the 9 targets to determine the molecular typing of SARS-CoV-2.
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| CN113764032A (en) * | 2021-10-21 | 2021-12-07 | 北京安智因生物技术有限公司 | Fluorescent quantitative PCR platform gene intelligent identification and report automatic system |
| WO2023159572A1 (en) * | 2022-02-28 | 2023-08-31 | 江苏汇先医药技术有限公司 | Detection kit, primer composition and detection method for novel coronavirus sars-cov-2 |
| CN114807454A (en) * | 2022-06-29 | 2022-07-29 | 北京市疾病预防控制中心 | Primer composition for detecting mutation site of novel coronavirus |
| CN114807454B (en) * | 2022-06-29 | 2022-09-02 | 北京市疾病预防控制中心 | Primer composition for detecting mutation site of novel coronavirus |
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