CN111500768B - Primer probe for identifying novel coronavirus and application of primer probe in dual-digital PCR - Google Patents
Primer probe for identifying novel coronavirus and application of primer probe in dual-digital PCR Download PDFInfo
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Abstract
The invention discloses a dual digital PCR primer, a probe, an identification method and an identification kit for screening and identifying novel coronavirus (SARS-CoV 2) and other SARS coronaviruses. The invention designs a first set of primers and probes (marked as FAM) according to the specific region of the S gene sequence of SARS-CoV2 virus (different from other SARS viruses), designs a second set of primers and probes (marked as HEX) according to the gene sequence of the SARS-CoV2 virus conserved region E, overcomes the defect of high false positive rate of the qPCR nucleic acid detection method for determining novel coronavirus recommended by WHO and CDC in China at present, has the advantages of high sensitivity, accurate quantification and effective differentiation of the novel coronavirus and other SARS viruses, and can meet the requirements of accurate molecular identification 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 screening and identifying primer, a probe, an identifying method and an identifying kit for screening and identifying novel coronavirus and other SARS coronavirus.
Background
At present, the national CDC and WHO recommend fluorescence quantitative PCR as a novel coronavirus (SARS-CoV 2) diagnosis confirming method, however, a large number of false negative results are found in the practical clinical application process, and the problems of the fluorescence PCR method cannot be ignored except for factors such as a virus RNA nucleic acid extraction method, a sampling part and nucleic acid degradation caused by improper storage. Compared with fluorescent quantitative PCR, the digital PCR has the advantages of high sensitivity and accurate quantification; in addition, no method for identifying and distinguishing the novel coronavirus from other SARS viruses simultaneously exists at present, which has great significance for epidemic disease prevention and control and screening of other similar diseases.
Disclosure of Invention
The invention provides a primer and a probe for identifying a novel coronavirus (SARS-CoV 2), wherein the primer and the probe comprise a forward primer NCS-F, a reverse primer NCS-R and a probe sequence NCS-P;
the sequence of the forward primer NCS-F is as follows:
5’TGGTGCAGGTATATGCGCTA3’；
the sequence of the reverse primer NCS-R is as follows:
5’GTGACATAGTGTAGGCAATGATG3’；
the sequence of the probe sequence NCS-P is as follows:
5’AGACTCAGACTAATTCTCCTCGGCGG-3’。
in one embodiment, the primers and probes for identifying a novel coronavirus (SARS-CoV 2) further comprise a forward primer NCE-F, a reverse primer NCE-R, and a probe sequence NCE-P;
the sequence of the forward primer NCE-F is as follows:
5’AGGTACGTTAATAGTTAATAGCGTAC 3’；
the sequence of the reverse primer NCE-R is as follows:
5’CAATATTGCAGCAGTACGCACA 3’；
the sequence of the probe sequence NCE-P is as follows:
5’CACTAGCCATCCTTACTGCGCTTCGA-3。
in one embodiment, the probe sequence NCS-P has a first fluorescent reporter at one end and a first fluorescent quencher at the other end; one end of the probe sequence NCE-P is provided with a second fluorescence reporter group, and the other end is provided with a second fluorescence quencher group.
In one embodiment, the first and second fluorescent reporter groups are selected from FAM, VIC, HEX, CY5, or ROX; the first fluorescence quenching group and the second fluorescence quenching group are selected from BHQ1, BHQ2, eclipse or TAMRA.
Preferably, the first fluorescent reporter group is arranged at the 5 'end of the probe sequence NCS-P, and the first fluorescent quencher group is arranged at the 3' end of the probe sequence NCS-P; the second fluorescence reporter group is arranged at the 5 'end of the probe sequence NCE-P, and the second fluorescence quencher group is arranged at the 3' end of the probe sequence NCE-P.
In a preferred embodiment, the first and second fluorescent reporter groups are different; preferably, the first fluorescent reporter is FAM and the second fluorescent reporter is HEX.
On the other hand, the invention also provides the application of the primer and the probe in identifying SARS-Cov2 virus and non SARS-Cov2 coronavirus.
Further, the application comprises the steps of carrying out double digital PCR on a sample to be detected by utilizing a forward primer NCS-F, a reverse primer NCS-R, a probe sequence NCS-P, the forward primer NCE-F, the reverse primer NCE-R and the probe sequence NCE-P; when the first fluorescence reporter group and the second fluorescence reporter group are both positive, the sample to be detected is SARS-Cov2 virus; when the first fluorescence reporter group is negative and the second fluorescence reporter group is positive, the sample to be detected is non-SARS-Cov 2 coronavirus; and when the first fluorescent reporter group and the second fluorescent reporter group are negative, the sample to be detected is not coronavirus.
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 double digital PCR reaction kit.
Furthermore, the type of the sample to be detected can be judged according to the reaction result; specifically, when the first fluorescence reporter group and the second fluorescence reporter group are both positive, the sample to be detected is SARS-Cov2 virus; when the first fluorescence reporter group is negative and the second fluorescence reporter group is positive, the sample to be detected is non-SARS-Cov 2 coronavirus; and when the first fluorescent reporter group and the second fluorescent reporter group are negative, the sample to be detected is not coronavirus.
Further, the non-SARS-Cov 2 coronavirus includes SARS virus and MERS virus.
The primer and the probe for identifying the novel coronavirus (SARS-Cov 2) provided by the invention have low detection sensitivity, can distinguish and identify the novel coronavirus, other SARS viruses except the SARS-Cov2 virus and other non-related pathogens by a double digital PCR reaction, and have wide application prospect.
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FIG. 1. Dual digital PCR assay identifies novel coronavirus susceptibility assays. From left to right, there are 8 wells, 1:10 7 copies/interaction (including E gene and S gene); 2:10 6 copies/reaction；3：10 5 copies/reaction；4：10 4 copies/reaction；5：10 3 copies/reaction；6：10 2 copies/reaction；7：10 1 copies/reaction；8：Negative control.
FIG. 2. Dual droplet digital PCR assay for identifying novel coronavirus specificity assays. Ch1 is FAM fluorescence signal, and Ch2 is HEX fluorescence signal. From left to right, there are 8 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. 3 shows the result of double micro-drop digital PCR identification and detection of different SARS viruses. From left to right, 4 wells in total, from left to right, in order, novel coronavirus, nucleic acid mimicking SARS virus, PEDV virus, and negative control.
FIG. 4 shows the detection results of the double-droplet digital PCR clinical samples. Ch1 is FAM fluorescence signal, and Ch2 is HEX fluorescence signal. 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 is further described with reference to the following examples, which are intended to be illustrative of the preferred embodiments of the invention only, and not to be limiting of the invention in any way. 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.
1. Obtaining of primer and probe sequence and construction of positive plasmid
Based on the full-length nucleic acid sequence of the novel coronavirus (SARS-CoV 2), the comparison analysis is carried out, and the comparison with the full-length sequences of SARS virus and SARS-like virus is carried out to find out the specific sequence S gene of SARS-CoV2 virus and the conserved sequence E gene of SARS virus, and then the primer and probe are designed based on the above sequences.
The primer probes involved are as follows:
this example completes the sensitivity test of the digital PCR method by constructing a standard plasmid. Based on the full-length sequences of the E gene and the S gene of SARS-CoV2, the full-length E gene and the full-length S gene are synthesized based on the pUC57 plasmid, and a recombinant plasmid is constructed. Transferring into competent cells of Escherichia coli (Escherichia coli) by heat shock method, coating plate to select monoclonal colony, propagating and culturing the colony, and extracting plasmid from bacterial liquid. After quantification using Nanodrop, plasmid concentrations were calculated and then diluted to 5 × 10 respectively 6 copies/μl，5×10 5 copies/μl，5×10 4 copies/μl，5×10 3 copies/μl，5×10 2 copies/. Mu.l, 5X 10 copies/. Mu.l, 5 copies/. Mu.l. Can be used for subsequent sensitivity test.
2. And (3) processing an actual detection sample and extracting a virus RNA genome.
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 swab of 1 patient in 1ml of normal saline, shaking, mixing, centrifuging at 5000r/min for 10min, and collecting 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 detailed procedure was as follows (1) adding 0.9ml of guanidinium isocyanate-glass powder lysis solution (4.7 mol/L guanidinium isothiocyanate, 20mmol/L LEDTA,100mmol/L Tris-HCl pH6.4,1% Triton X-100 and 10g glass powder) to 0.1ml of the sample solution, mixing well, working at room temperature for 1min, centrifuging at 6 × g for 1min. (2) The supernatant was discarded, and 1ml of a guanidine isothiocyanate rinse (4.7 mol/L guanidine isothiocyanate and 100mmol/L Tris-HCl pH 6.4) was added thereto, followed by mixing and centrifugation at 6000 Xg for 1min. (3) The supernatant was discarded, 1ml of 70% ethanol was added, and the mixture was centrifuged at 6000 Xg for 1min. (4) Abandoning 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 and mixing uniformly, centrifuging for 1min at 6000 Xg, and taking supernatant as a template of digital PCR.
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. And (2) adding 560. Mu.l of absolute ethyl alcohol. (3) Mixing, adding into filter column twice, adding 630 μ l each time, and centrifuging at 8000r/min for 1min. (4) adding 500. Mu.l of AW1 solution, and centrifuging at 8000r/min for 1min. (5) adding 500. Mu.l of AW2 solution, and centrifuging for 3min at 14000 r/min. (6) Finally, 60 mul AVE solution is added, RNA is eluted at 8000r/min for 1min and is used as a template for PCR amplification.
3. Performing a droplet digital PCR:
the microdroplet digital PCR reaction comprises 4 steps of preparing the system, generating microdroplets, amplifying cycles and reading signals. RT-ddPCR was performed using the prepared RNA or (DNA plasmid) as a template in a 20. Mu.L system recommended by One-step RT-dd RT-PCR Kit for Probes Kit. The RT-ddPCR reaction was performed as soon as possible within 30min after the membrane was sealed or within 4h in a 4 ℃ freezer. RT-ddPCR reaction (20. Mu.L): supermix 5. Mu.L, reverse transcriptase 2. Mu.L, 300mM DTT 1. Mu.L, upstream primers NCS-F, NCE-F each 0.8. Mu.L (10. Mu. Mol/L), downstream primers NCS-R, NCE-R each 0.8. Mu.L (10. Mu. Mol/L), probes NCS-P, NCE-P each 0.4. Mu.L (10. Mu. Mol/L), RNA template 2. Mu.L, RNase-free Water 6. Mu.L. The reaction conditions were set as follows: 60min at 42 ℃; 10min at 95 ℃; 30s at 95 deg.C, 1min at 56 deg.C for 40 cycles; 10min at 98 ℃; infinity at 4 ℃. And after the RT-ddPCR reaction is finished, putting the 96-well plate into a QX200 Droplet Reader, sequentially inputting sample information, running, automatically acquiring fluorescent signals by an instrument, and calculating a final result by adopting software Quanta Soft.
Digital PCR sensitivity test: RT-ddPCR reaction (20. Mu.L): supermix 5. Mu.L, reverse transcriptase 2. Mu.L, 300mM DTT 1. Mu.L, upstream primers NCS-F, NCE-F each 0.8. Mu.L (10. Mu. Mol/L), downstream primers NCS-R, NCE-R each 0.8. Mu.L (10. Mu. Mol/L), probes NCS-P, NCE-P each 0.4. Mu.L (10. Mu. Mol/L), DNA plasmid template E gene and S gene each 2. Mu.L (seven concentration gradients in 10-fold dilution total, from 5 to 510 6 copies/. Mu.l to 5 copies/. Mu.l), RNase-free Water 4. Mu.L.
Digital PCR specificity test: RT-ddPCR reaction (20. Mu.L): supermix 5. Mu.L, reverse transcriptase 2. Mu.L, 300mM DTT 1. Mu.L, upstream primers NCS-F, NCE-F each 0.8. Mu.L (10. Mu. Mol/L), downstream primers NCS-R, NCE-R each 0.8. Mu.L (10. Mu. Mol/L), probes NCS-P, NCE-P each 0.4. Mu.L (10. Mu. Mol/L), viral nucleic acids each 2. Mu.L (total nucleic acid amount between 50 and 100 ng), RNase-free Water 6. Mu.L. 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.
Detection distinguishes SARS coronavirus (SARS-Cov) from emerging coronavirus (SARS-Cov 2): RT-ddPCR reaction (20. Mu.L): supermix 5. Mu.L, reverse transcriptase 2. Mu.L, 300mM DTT 1. Mu.L, upstream primers NCS-F, NCE-F each 0.8. Mu.L (10. Mu. Mol/L), downstream primers NCS-R, NCE-R each 0.8. Mu.L (10. Mu. Mol/L), probes NCS-P, NCE-P each 0.4. Mu.L (10. Mu. Mol/L), viral nucleic acids each 2. Mu.L (total nucleic acid amount between 50 and 100 ng), RNase-free Water 6. Mu.L. Artificially synthesizing the full-length sequence of S gene of SARS coronavirus, and constructing control plasmid P-SARS-S based on pUC57 plasmid. The plasmid P-SARS-S and new coronavirus E gene plasmid are used as template to simulate SARS virus genome and perform double digital PCR detection and distinguishing experiment of new coronavirus and other SARS coronavirus. Four groups: positive controls (SARS-Cov 2 nucleic acid reverse transcription cDNA), mock SARS virus (plasmid P-SARS-S and plasmid E gene), PEDV coronavirus nucleic acid and RNase-free water negative controls, respectively.
4. 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. Digital PCR reaction (20 μ L): supermix 5. Mu.L, reverse transcriptase 2. Mu.L, 300mM DTT 1. Mu.L, upstream primers NCS-F, NCE-F each 0.8. Mu.L (10. Mu. Mol/L), downstream primers NCS-R, NCE-R each 0.8. Mu.L (10. Mu. Mol/L), probes NCS-P, NCE-P each 0.4. Mu.L (10. Mu. Mol/L), clinical specimen nucleic acids each 2. Mu.L (total nucleic acid amount between 50 and 100 ng), RNase-free Water 6. Mu.L. The positive control is new coronavirus nucleic acid reverse transcription cDNA.
5. And (3) test results:
respectively carrying out double digital PCR by using two groups of primer probes, wherein the first group of primer probes are NCS-F, NCS-R, NCS-P, NCE-F, NCE-R and NCE-P; the second group of primer probes are NCS-F1, NCS-R1, NCS-P1, NCE-F1, NCE-R1 and NCE-P1. The sensitivity of detection using the second set of primer probes is very low, even 10 3 Neither copies/reaction template gave a positive result and was discarded. The sensitivity of the first set of primer probes can be within 10 copies. As shown in FIG. 1, when double digital PCR was carried out using two sets of primer probes of the first group, NCS-F, NCS-R, NCS-P, NCE-F, NCE-R and NCE-P, the detection limits were all within 10copies, see FIG. 1, second right row, upper panel of FIG. 1, FAM-labeled primer probes for detecting the S gene, and lower panel HEX-labeled primer probes for detecting the E gene.
As shown in figure 2, the first group of primer probes has good detection result specificity, the two groups of primer probes have no cross reaction with PEDV, TGEV, PPRV, FMDV, RV and PPV, FAM and HEX fluorescent signals are only in a novel coronavirus column, the first column on the right shows positive, and the rest are negative.
As shown in FIG. 3, double digital PCR using the first set of primer probes can be used to distinguish and identify novel coronaviruses and other SARS-like viruses; aiming at the nucleic acid simulating SARS virus, only the HEX channel of the digital PCR shows positive, and the FAM channel is negative; but is double-channel positive for the novel coronavirus; PEDV was double negative for the unrelated coronavirus.
In actual use, the detection coincidence rate of the clinical samples is 100%, as shown in fig. 4, the detection results of the two sets of primer probes in the first group are negative for 2011 and 2013 clinical samples, and positive for 2012, 2014, 2015 and 2016 clinical samples, so as to obtain the certificate issued by the organization of the clinical testing center in Shanghai city.
Claims (11)
1. Primers and probes for identifying a novel coronavirus (SARS-CoV 2), the primers and probes comprising a forward primer NCS-F, a reverse primer NCS-R and a probe sequence NCS-P, and a forward primer NCE-F, a reverse primer NCE-R and a probe sequence NCE-P; it is characterized in that the preparation method is characterized in that,
the sequence of the forward primer NCS-F is as follows:
5’TGGTGCAGGTATATGCGCTA3’；
the sequence of the reverse primer NCS-R is as follows:
5’GTGACATAGTGTAGGCAATGATG3’；
the sequence of the probe sequence NCS-P is as follows:
5’AGACTCAGACTAATTCTCCTCGGCGG-3’；
the sequence of the forward primer NCE-F is as follows:
5’AGGTACGTTAATAGTTAATAGCGTAC 3’；
the sequence of the reverse primer NCE-R is as follows:
5’CAATATTGCAGCAGTACGCACA 3’；
the sequence of the probe sequence NCE-P is as follows:
5’CACTAGCCATCCTTACTGCGCTTCGA-3。
2. the primers and probe for identifying a novel coronavirus (SARS-CoV 2) according to claim 1, wherein the probe sequence NCS-P has a first fluorescent reporter group at one end and a first fluorescent quencher group at the other end; one end of the probe sequence NCE-P is provided with a second fluorescence reporter group, and the other end is provided with a second fluorescence quencher group.
3. The primers and probes for identifying novel coronaviruses (SARS-CoV 2) according to claim 2, wherein the first and second fluorescent reporter groups are selected from FAM, VIC, HEX, CY5 or ROX; the first fluorescence quenching group and the second fluorescence quenching group are selected from BHQ1, BHQ2, eclipse or TAMRA.
4. The primers and probe for identifying a novel coronavirus (SARS-CoV 2) according to claim 3, wherein the first fluorescent reporter group is placed at the 5 'end of the probe sequence NCS-P and the first fluorescent quencher group is placed at the 3' end of the probe sequence NCS-P; the second fluorescent reporter group is arranged at the 5 'end of the probe sequence NCE-P, and the second fluorescent quencher group is arranged at the 3' end of the probe sequence NCE-P.
5. The primers and probe for identifying a novel coronavirus (SARS-CoV 2) according to claim 4, wherein the first and second fluorescent reporter groups are different.
6. The primers and probe for identifying a novel coronavirus (SARS-CoV 2) according to claim 5, wherein the first fluorescent reporter group is FAM and the second fluorescent reporter group is HEX.
7. Use of the primers and probes for identifying a novel coronavirus (SARS-CoV 2) as claimed in any one of claims 1 to 6 for the preparation of a kit for identifying SARS-Cov2 virus and/or non SARS-Cov2 coronavirus.
8. The use according to claim 7, wherein the virus type of the sample to be tested is judged according to the following reaction result of the kit:
when the first fluorescence reporter group and the second fluorescence reporter group are both positive, the sample to be detected is SARS-Cov2 virus;
when the first fluorescence reporter group is negative and the second fluorescence reporter group is positive, the sample to be detected is non-SARS-Cov 2 coronavirus;
and when the first fluorescent reporter group and the second fluorescent reporter group are negative, the sample to be detected is not coronavirus.
9. Use according to claim 7 or 8, characterized in that the kit is a dual digital PCR reaction kit.
10. A kit for identifying SARS-Cov2 virus and/or non-SARS-Cov 2 coronavirus, comprising the primers and probes for identifying novel coronavirus (SARS-Cov 2) of any one of claims 1 to 6.
11. The kit of claim 10, wherein the kit is a dual digital PCR reaction kit.
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