CN111235316A - Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA - Google Patents
Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA Download PDFInfo
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Abstract
The invention discloses a triple RPA primer, a probe, an identification method and an identification kit for rapidly screening and detecting novel coronavirus (SARS-CoV2) and other SARS coronaviruses. The invention designs a first set of primers and probes (marked as FAM) in the N gene sequence region of SARS-CoV2 virus; designing a second set of primers and probes (marked as HEX) in the conserved region E gene sequence of the SARS virus-like virus; a third set of primers and probes (labeled Cy5, different from other SARS viruses) was designed in the S gene sequence specific region of SARS-CoV2 virus. Compared with the qPCR nucleic acid detection method for definite diagnosis of the novel coronavirus recommended by WHO and national CDC at present, the method has the advantages of high sensitivity, rapidness and effective differentiation of the novel coronavirus and other SARS viruses, can shorten the nucleic acid detection time limit of the virus from 90min to 20min, and can meet the rapid screening requirements of the novel coronavirus and other SARS viruses.
Description
Technical Field
The invention belongs to the field of applied biotechnology, and particularly relates to a specific rapid screening detection primer, a probe, an identification method and an identification kit for screening and identifying novel coronavirus and other SARS coronavirus.
Background
Pneumonia epidemic situation (COVID-19) caused by novel coronavirus (SARS-CoV2) is rolling around the world at present, and has great influence on the normal life of economy, society and people, and the rapid diagnosis of new coronavirus cases becomes an important means for cutting off infection sources and controlling the spread of epidemic situation. Especially, the current domestic epidemic situation tends to be stable, the foreign epidemic situation is severe, the number of input disease cases is increased, and the requirement on the accuracy of nucleic acid investigation is higher.
At present, the national CDC and WHO recommend fluorescence quantitative PCR as a novel coronavirus diagnosis method, however, the timeliness is not particularly good in the practical application process, the reverse transcription fluorescence PCR takes 90min, and a special fluorescence PCR instrument is required for data analysis. Compared with fluorescent quantitative PCR, the fluorescent constant-temperature RPA has the advantages of rapidness, accuracy, low requirements on instruments and equipment and the like; in addition, the triple RPA can be used for identifying and distinguishing the novel coronavirus from other SARS viruses, which has great influence on epidemic disease prevention and control and screening other similar epidemic diseases.
Disclosure of Invention
The invention provides a group of primers and probes for identifying a novel coronavirus (SARS-CoV2), wherein the primers and probes comprise a first set of primer probes, the first set of primer probes comprise a forward primer NF, a reverse primer NR and a probe NP, and the sequences of the primers and probes are as follows:
forward primer NF:
AAGACCTTAAATTCCCTCGAGGACAAGGCGTTC；
reverse primer NR:
AGGTAGTAGAAATACCATCTTGGACTGAGATCT3’；
the probe NP:
CACCAATAGCAGTCCAGATGACCAAATTGGC/i6FAMdT/A/idSp//iBHQ1dT/ACCGAAGAGCTACCAG/iSpC3/。
wherein the fluorescence label of the probe NP is FAM.
Further, the primer and probe set for identifying the novel coronavirus (SARS-CoV2) further comprises a second set of primer and probe, wherein the second set of primer and probe comprises a forward primer EF, a reverse primer ER and a probe EP, and the sequence of the primer and probe is as follows:
forward primer EF:
GACAGGTACGTTAATAGTTAATAGCGTACTTCT；
reverse primer ER:
AGACTCACGTTAACAATATTGCAGCAGTACGCA；
the probe EP:
TTCGTGGTATTCTTGCTAGTTACACTAGCCA/iHEXdT/C/idSp//iBHQ1dT/TACTGCGCTTCGATTG/iSpC3/；
wherein the fluorescent label of the probe EP is HEX.
Further, the primer and probe set for identifying the novel coronavirus (SARS-CoV2) further comprises a third set of primer and probe, wherein the third set of primer and probe comprises a forward primer SF, a reverse primer SR and a probe SP, and the sequences of the primer and probe are as follows:
forward primer SF:
AACCATTGGTAGATTTGCCAATAGGTATTAACAT；
reverse primer SR:
CCTAGGTTGAAGATAACCCACATAATAAGCTGC；
and (3) a probe SP:
TACATAGAAGTTATTTGACTCCTGGTGATTCT/iCy5dT/C/idSp//iBHQ2dT/CAGGTTGGACAGCTG/iSpC3/；
the fluorescent label of the probe SP was Cy 5.
On the other hand, the invention also provides the application of the primer and the probe in identifying SARS-Cov2 virus and non-SARS-Cov 2 coronavirus.
Further, the application comprises the step of carrying out triple fluorescence RPA reaction on a sample to be detected by using the primer and the probe; if the probe NP, the probe EP and the probe SP are all positive, judging the coronavirus as a novel coronavirus; if the probe EP is positive and the probe NP and the probe SP are negative, the coronavirus is judged to be non-SARS-Cov 2 coronavirus; if all of the probe NP, the probe EP and the probe SP are negative, the non-coronavirus is judged.
On the other hand, the invention also provides the application of the primer and the probe in preparing a kit for identifying SARS-Cov2 virus and non-SARS-Cov 2 coronavirus.
Further, the kit is a triple fluorescence RPA reaction kit.
Furthermore, the type of the sample to be detected can be judged according to the reaction result; specifically, if the probe NP, the probe EP, and the probe SP are all positive, the coronavirus is judged to be a novel coronavirus; if the probe EP is positive and the probe NP and the probe SP are negative, the coronavirus is judged to be non-SARS-Cov 2 coronavirus; if all of the probe NP, the probe EP and the probe SP are negative, the non-coronavirus is judged.
Further, the non-SARS-Cov 2 coronavirus includes SARS virus and MERS virus.
On the other hand, the invention also provides a specific triple fluorescence RPA kit which can be used for rapidly screening and detecting the novel coronavirus (SARS-Cov2) and other coronaviruses, and the kit comprises an RNA extraction reagent, the primer and the probe.
The primer and the probe for identifying the novel coronavirus (SARS-Cov2) provided by the invention have low detection sensitivity, can distinguish and identify the novel coronavirus, other SARS viruses except SARS-Cov2 virus and other non-related pathogens by a triple fluorescence RPA reaction, and have wide application prospect.
Drawings
FIG. 1 shows the first set of primer probes, target N gene, for triple fluorescence RPA detection of novel coronavirus susceptibility test. From left to right, there are 7 wells, 1: 106copies/reaction (N gene); 2: 105copies/reaction；3：104copies/reaction；4：103copies/reaction；5：102copies/reaction；6：101copies/reaction；7：Negative control。
FIG. 2 shows a second set of primer probes, target E gene, for triple fluorescent RPA detection of novel coronavirus susceptibility assays. From left to right, there are 7 wells, 1: 106copies/reaction (E gene); 2: 105copies/reaction；3：104copies/reaction；4：103copies/reaction；5：102copies/reaction；6：101copies/reaction；7：Negative control。
FIG. 3 shows the third set of primer probes, target S gene, for triple fluorescence RPA detection of susceptibility of novel coronavirus. From left to right, there are 7 wells, 1: 106copies/reaction (S gene); 2: 105copies/reaction；3：104copies/reaction；4：103copies/reaction；5：102copies/reaction；6：101copies/reaction；7：Negative control。
FIG. 4, triple fluorescent RPA assay for the detection of novel coronavirus specificity. From left to right, there are 7 wells, 1: PEDV virus nucleic acid; 2: TGEV viral nucleic acid; 3: PPRV viral nucleic acid; 4: FMDV viral nucleic acids; 5: an RV viral nucleic acid; 6: a PPV viral nucleic acid; 7: negative control; 8: positive control (nCov-cDNA).
FIG. 5, triple fluorescent RPA detection of mock SARS virus nucleic acid. Three groups were tested simultaneously, positive control, negative control, PEDV. Only the results of nucleic acid detection of the SARS-simulating virus were shown, and the other three groups showed similar results to those in FIG. 4, and were not repeated.
FIG. 6, results of triple fluorescent RPA clinical sample detection (FAM signal, N gene). From left to right, there are 7 wells, 1: clinical samples 2011; 2: a clinical specimen 2012; 3: clinical specimen 2013; 4: clinical sample 2014; 5: clinical sample 2015; 6: a clinical sample 2016; 7: positive control (nCov-cDNA).
Fig. 7, results of triple fluorescent RPA clinical sample detection (HEX signal, E gene). From left to right, there are 7 wells, 1: clinical samples 2011; 2: a clinical specimen 2012; 3: clinical specimen 2013; 4: clinical sample 2014; 5: clinical sample 2015; 6: a clinical sample 2016; 7: positive control (nCov-cDNA).
Fig. 8 shows the results of detection of triple fluorescent RPA clinical samples (Cy5 signal, S gene). From left to right, there are 7 wells, 1: clinical samples 2011; 2: a clinical specimen 2012; 3: clinical specimen 2013; 4: clinical sample 2014; 5: clinical sample 2015; 6: a clinical sample 2016; 7: positive control (nCov-cDNA).
Detailed description of the preferred embodiments
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 obtaining of primer and Probe sequences and construction of Positive plasmids
Comparing and analyzing the full-length nucleic acid sequence of the novel coronavirus (SARS-CoV2), comparing the full-length nucleic acid sequence with the full-length sequences of SARS virus and SARS-like virus to find out the specific sequence S gene of SARS-CoV2 virus, the conserved sequence N gene of virus, the conserved sequence E gene of SARS virus, the gene Orf1Ab and the gene M, and then designing primers and probes according to the sequences; the primers and probes referred to in the present application are shown in the following table:
the sensitivity test of the fluorescent RPA method is completed by constructing a standard plasmid. Based on the pUC57 plasmid, the target gene was synthesized and a recombinant plasmid was constructed. Transformation into E.coli (Escherichia coli) Using Heat shock methodhia coli) competent cells, coating a flat plate to select a monoclonal colony, carrying out propagation culture on the colony, and extracting plasmid from a bacterial liquid. After quantification using Nanodrop, plasmid concentrations were calculated and then diluted to 5 × 10 respectively6copies/μl，5×105copies/μl，5×104copies/μl，5×103copies/μl，5×102copies/. mu.l, 5X 10 copies/. mu.l, 5 copies/. mu.l. Can be used for subsequent sensitivity test.
Example 2 treatment of actual test samples and extraction of viral RNA genomes
The test sample includes, but is not limited to, human serum, plasma, whole blood, stool, throat swab, sputum, etc. Sample treatment: dissolving 1g of feces or throat swabs of 1 patient in 1ml of physiological saline, shaking and mixing uniformly, centrifuging at 5000r/min for 10min, and taking supernatant to extract virus RNA. Adding 10g/L acetylcysteine into the sputum, shaking for 1h at room temperature, and fully liquefying to extract virus RNA. Anticoagulant (heparin, etc.) should be added to the blood plasma and whole blood.
Extracting virus RNA by a guanidinium isothiocyanate-glass powder method: the specific operation steps are (1) adding 0.9ml of guanidinium isocyanate-glass powder lysate (4.7mol/L guanidinium isothiocyanate, 20mmol/L LEDTA,100mmol/L Tris-HClpH6.4, 1% Triton X-100 and 10g of glass powder) into 0.1ml of sample solution, uniformly mixing, acting at room temperature for 10min, and centrifuging at 6000 Xg for 1 min. (2) The supernatant was discarded, and 1ml of a guanidine isothiocyanate rinse (4.7mol/L guanidine isothiocyanate and 100mmol/L Tris-HCl pH6.4) was added thereto, followed by mixing and centrifugation at 6000 Xg for 1 min. (3) The supernatant was discarded, 1ml of 70% ethanol was added, and the mixture was centrifuged at 6000 Xg for 1 min. (4) And (4) discarding the supernatant, inverting the plastic centrifuge tube, and baking for 10-15 min at 37 ℃. (5) Adding 50 mul of DEPC treated deionized water to elute RNA, shaking, mixing uniformly, centrifuging at 6000 Xg for 1min, and taking supernatant as a template of fluorescent RPA.
Viral RNA was extracted using QIAGEN's kit (products from other reagents were also used): the operation is carried out according to the instruction, and the specific operation steps are (1) 560 mul of AVL is added into a 1.5ml plastic centrifuge tube, then 140 mul of specimen liquid is added, and the mixture is evenly mixed and acted for 10min at room temperature. (2) 560. mu.l of absolute ethanol was added. (3) Mixing, adding into filter column twice, adding 630 μ l each time, and centrifuging at 8000r/min for 1 min. (4) Add 500. mu.l AW1 solution and centrifuge at 8000r/min for 1 min. (5) Add 500. mu.l AW2 solution and centrifuge for 3min at 14000 r/min. (6) Finally, 60 mul AVE liquid is added, RNA is eluted at 8000r/min for 1min and is used as a template for PCR amplification.
Example 3 implementation of triple fluorescent RPA
The triple fluorescence RPA reaction comprises 2 steps of preparing a system, amplifying and reading signals. A50 mu L system recommended by an RNA constant-temperature rapid amplification kit (fluorescent type) is adopted, prepared RNA or DNA plasmid is taken as a template, the sequences of the used primers and probes are summarized, and the triple fluorescent RPA reaction is carried out. Fluorescent RPA reaction system (50 μ L): buffer A29.4. mu.L, forward primers NF, EF, SF 0.8. mu.L (10. mu. mol/L), reverse primers NR, ER, SR 0.8. mu.L (10. mu. mol/L), probes NP, EP, SP 0.3. mu.L (10. mu. mol/L), nucleic acid template 2. mu.L, RNase-free Water 10.2. mu.L, ROX dye 0.2. mu.L, Buffer B2.5. mu.L. The reaction conditions were set as follows: fluorescence signals were collected every 30S for 40 cycles at 42 ℃ for 20 min. The instrument automatically collects fluorescent signals, can observe fluorescent amplification signals in real time, and is a 7500 fluorescent PCR instrument of ABI.
Fluorescence RPA sensitivity test: the same procedure as in the above-mentioned system was repeated except that the volume of the system was 50. mu.L. DNA plasmid template N gene, E gene and S gene of the same concentration 2 uL each (10 times dilution total six concentration gradient, from 5X 106copies/. mu.l to 5 copies/. mu.l), negative control RNase-free Water 2. mu.L.
Fluorescent RPA specificity test: reaction system (50. mu.L), wherein each viral nucleic acid 2. mu.L (total nucleic acid amount between 50 and 100 ng). Specific detection includes 6 viruses, two of which are PEDV (porcine epidemic diarrhea virus), TGEV (transmissible gastroenteritis virus), RNA virus PPRV (Peste des petits ruminants virus), RNA virus FMDV (foot and mouth disease virus), RNA virus RV (rotavirus) and DNA virus PPV (porcine parvovirus). The positive control is new coronavirus nucleic acid reverse transcription cDNA, and the negative control is RNase-free water.
The detection distinguishes SARS coronavirus (SARS-Cov) from novel coronavirus (SARS-Cov 2): reaction system (50. mu.L). Artificially synthesizing the full-length S gene sequence of SARS coronavirus and the full-length N gene of SARS virus, and constructing control plasmids P-SARS-S and P-SARS-N based on pUC57 plasmid. The plasmid P-SARS-S, P-SARS-N and new coronavirus E gene are used as template to simulate SARS virus genome and develop the experiment of triple RPA detection to distinguish new coronavirus and other SARS coronavirus. Four groups are used, wherein positive control is new coronavirus nucleic acid reverse transcription cDNA, negative control is RNase-freewater, other coronavirus PEDV and SARS coronavirus mimic nucleic acid.
And (3) detecting a clinical sample: taking part in a novel examination plan for detecting and examining nucleic acid of coronavirus from the organization of clinical laboratory center in Shanghai, 6 inactivated clinical samples with the batch numbers of 2011, 2012, 2013, 2014, 2015 and 2016 are obtained, and the sample amount is 600 muL/sample. The RNA nucleic acid was extracted from a sample of 50. mu.L/piece in the same manner as described above, and the nucleic acid was dissolved in 20. mu.L of RNase-free water. The RPA reaction (50. mu.L) was performed as described above, with 2. mu.L of nucleic acid in each clinical sample (total amount of nucleic acid between 50 and 100 ng). The positive control is new coronavirus nucleic acid reverse transcription cDNA, and the negative control is RNase-free water.
Example 4, detection results:
the primers and probes referred to in example 1 were tested for sensitivity and specificity. In a triple RPA reaction (requiring three primer probes), the primer probe combinations used were selected as follows:
firstly, the inventor carries out analysis and comparison on the primer probe combination of NF/NR/NP, NF2/NR2/NP2, ORF/ORR/ORP, ORF2/ORR2/ORP2 and MF/MR/MP, and the combination sensitivity of the primer probe combination of NF2/NR2/NP2, MF/MR/MP and ORF/ORR/ORP is 103The sensitivity is lower in the order of copies/interaction, and the negative control of the combination of ORF2/ORR2/ORP2 primer probe has nonspecific amplification, so that the first set of primer probe of the triple RPA reaction selects NF/NR/NP.
Two groups of primer probe combinations are designed aiming at the E gene, and through tests, the analysis sensitivity of the probe combination of EF2/ER2/EP2 is low and can only reach 104copies/reaction, so the second set of primer probes for the triple RPA reaction selects EF/ER/EP;
and aiming at the S gene, selecting an SF/SR/SP primer probe combination, and testing the combination to obtain the third set of primer probe SF/SR/SP for the triple RPA reaction, wherein the sensitivity and the specificity of the combination are good.
For the three sets of primer probes (SF/SR/SP, EF/ER/EP and NF/NR/NP) of the selected triple RPA reaction, the sensitivity test of the triple fluorescence RPA detection method shows that the detection limit (sensitivity) of the triple fluorescence RPA detection method to the N gene is 102copies/reaction (FIG. 1), the detection limit (sensitivity) for the E gene was also 102copies/reaction (FIG. 2), the detection limit (sensitivity) for the S gene is within 10copies (FIG. 3). In addition, the specificity of the primer probe is good, the fluorescent RPA method can detect the novel coronavirus, all the genes E, S and N are positive, and no cross reaction is caused with PEDV, TGEV, PPRV, FMDV, RV and PPV (figure 4); detecting SARS coronavirus mimic nucleic acid, the results show that the E gene is positive, and the S gene and N gene are negative (FIG. 5), confirming that the triple RPA detection method can be used for identifying and distinguishing new coronavirus from SARS-like virus; in the clinical sample detection of the triple RPA, the N gene detection result shows that three samples 2012, 2014 and 2015 are positive (fig. 6), the E gene detection result also shows that three samples 2012, 2014 and 2015 are positive (fig. 7), the S gene detection result shows that four samples 2012, 2014, 2015 and 2016 are positive (fig. 8), the coincidence rate of the S gene detection result in the triple RPA method is 100%, and the certificate issued by the clinical laboratory center in shanghai city is obtained.
Claims (8)
1. Primers and probes for identifying a novel coronavirus (SARS-CoV2), said primers and probes comprising a first set of primer probes comprising a forward primer NF, a reverse primer NR and a probe NP as follows:
forward primer NF:
AAGACCTTAAATTCCCTCGAGGACAAGGCGTTC；
reverse primer NR:
AGGTAGTAGAAATACCATCTTGGACTGAGATCT3’；
the probe NP:
CACCAATAGCAGTCCAGATGACCAAATTGGC/i6FAMdT/A/idSp//iBHQ1dT/ACCGAAGAGCTACCAG/iSpC3/。
2. the primers and probes for identifying a novel coronavirus (SARS-CoV2) according to claim 1, further comprising a second set of primer probes comprising a forward primer EF, a reverse primer ER and a probe EP as follows:
forward primer EF:
GACAGGTACGTTAATAGTTAATAGCGTACTTCT；
reverse primer ER:
AGACTCACGTTAACAATATTGCAGCAGTACGCA；
the probe EP:
TTCGTGGTATTCTTGCTAGTTACACTAGCCA/iHEXdT/C/idSp//iBHQ1dT/TACTGCGCTTCGATTG/iSpC3/。
3. the primers and probes for identifying a novel coronavirus (SARS-CoV2) according to claim 2, further comprising a third set of primer probes comprising a forward primer SF, a reverse primer SR and a probe SP as follows:
forward primer SF:
AACCATTGGTAGATTTGCCAATAGGTATTAACAT；
reverse primer SR:
CCTAGGTTGAAGATAACCCACATAATAAGCTGC；
and (3) a probe SP:
TACATAGAAGTTATTTGACTCCTGGTGATTCT/iCy5dT/C/idSp//iBHQ2dT/CAGGTTGGACAGCTG/iSpC3/。
4. use of the primers and probes for identifying a novel coronavirus (SARS-CoV2) according to any one of claims 1 to 3 in the preparation of a kit for identifying SARS-Cov2 virus and non-SARS-Cov 2 coronavirus.
5. The use according to claim 5, wherein the virus type of the sample to be tested is judged according to the following reaction result of the kit:
if the probe NP, the probe EP and the probe SP are all positive, judging that the sample to be detected is the novel coronavirus; if the probe EP is positive and the probe NP and the probe SP are negative, judging that the sample to be detected is non-SARS-Cov 2 coronavirus; and if the probe NP, the probe EP and the probe SP are all negative, judging that the sample to be detected is not coronavirus.
6. The use according to claim 4 or 5, wherein the kit is a triple fluorescent RPA reaction kit.
7. A specific triple fluorescent RPA kit for rapid screening and detection of novel coronavirus (SARS-Cov2) and other coronaviruses, which comprises the primers and probes for identifying novel coronavirus (SARS-Cov2) as claimed in any one of claims 1 to 3.
8. The kit of claim 7, further comprising an RNA extraction reagent.
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| CN112662811A (en) * | 2020-12-25 | 2021-04-16 | 北京美康基因科学股份有限公司 | Novel coronavirus 4 gene segment multiplex nucleic acid detection kit and application thereof |
| CN114410835A (en) * | 2021-12-02 | 2022-04-29 | 华南农业大学 | RPA-LFD kit for rapidly detecting novel coronavirus |
| CN114717363A (en) * | 2022-06-09 | 2022-07-08 | 中国科学院深圳先进技术研究院 | Instant nucleic acid detection method and detection kit for pathogenic mutant |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109072289A (en) * | 2016-03-04 | 2018-12-21 | 爱乐圣地亚哥公司 | Automate nido recombinase polymeric enzymatic amplification |
-
2020
- 2020-03-23 CN CN202010208673.1A patent/CN111235316B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109072289A (en) * | 2016-03-04 | 2018-12-21 | 爱乐圣地亚哥公司 | Automate nido recombinase polymeric enzymatic amplification |
Non-Patent Citations (6)
| Title |
|---|
| CORMAN VM ET AL: "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR", 《EURO SURVEILL》 * |
| KAZUYA SHIRATO ET AL: "Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan", 《 JPN.J.INFECT.DIS.》 * |
| RANA K. DAHER ET AL: "Recombinase Polymerase Amplification for Diagnostic Applications", 《CLINICAL CHEMISTRY》 * |
| 温旺荣等: "《临床分子诊断学（第二版）》", 31 March 2014, 广州科技出版社 * |
| 王越珉等: "新型冠状病毒及其检测方法研究进展", 《中国计量大学学报》 * |
| 郑泽宇等: "2019-nCoV肺炎的研究现状与未来展望", 《福建医药杂志》 * |
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