CN111378783A - Novel coronavirus 2019-nCoV nucleic acid kit and virus nucleic acid collection method - Google Patents
Novel coronavirus 2019-nCoV nucleic acid kit and virus nucleic acid collection method Download PDFInfo
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Abstract
The invention discloses a novel coronavirus 2019-nCoV nucleic acid kit and a virus nucleic acid acquisition method, wherein a specific primer and a fluorescent probe are designed for detecting novel coronavirus 2019-nCoV nucleic acid in a sample according to the novel coronavirus 2019-nCoV ORF1ab and N gene serving as amplification target regions; combining the prepared Super premix reaction liquid A with the Super premix reaction liquids B and C, performing Digital PCR absolute quantification on 2019-nCoV nucleic acid RNA by adopting a one-step method, and performing Digital PCR detection on diluted positive virus sample RNA. The invention has the beneficial effects that: through a novel virus nucleic acid acquisition method and a Digital PCR probe method, the nucleic acid positive detection rate of the novel coronavirus 2019-nCoV infection is improved, the process specification and the standard are ensured, the influence of manual and non-standard operation on the detection result is reduced, the reliability of the result is ensured, and meanwhile, the false negative result caused by the combination of the sampling of sputum and a throat swab and the detection of a real-time fluorescence PCR method is avoided.
Description
Technical Field
The invention relates to the field of detection kits, and mainly relates to a novel coronavirus 2019-nCoV nucleic acid kit and a virus nucleic acid acquisition method.
Background
2019novel coronaviruses (2019novel coronavirus, 2019-nCoV) were named by World Health Organization (WHO) in 12.1.2020. Reliable laboratory diagnosis is a primary task to facilitate public health interventions. In acute respiratory infections, real-time PCR, i.e. RT-PCR, is commonly used to detect pathogenic viruses in respiratory secretions.
At present, the real-time fluorescent PCR method is mainly adopted to detect 2019-nCoV nucleic acid (developing a reading frame 1ab and a nucleocapsid protein) clinically to carry out early diagnosis on a case, and the diagnosis can be confirmed only if the case is positive. The samples from which they were derived included primarily sputum, as well as nasal and pharyngeal swabs with or without viral transport media. For some samples with low abundance of viral load, the fluorescence PCR method often has many false negative results due to the sensitivity, and there are many reports in practical clinical application, and the samples often need to be repeatedly sampled for 2-3 times, even patients who have positive definite diagnosis only by the 6 th pharyngeal swab nucleic acid detection, which not only increases the workload of medical staff, but also the false negative patients are easy to become infectious sources again if the false negative patients are not properly treated. Cause the low reason of detectable rate, except the limitation of fluorescence PCR method itself, more importantly because the characteristic of novel coronavirus itself, cause upper respiratory tract virus low degree such as pharynx very low, consequently we need a more effective virus sample collection divides the method, combines the absolute number that Digital PCR can direct detection nucleic acid molecule simultaneously, and its sensitivity can be down to the characteristics that single copy can be accurate carry out quantitative determination to low abundance sample to can expect to improve the detectable rate greatly. Based on the current epidemiological investigation, the incubation period of the novel coronavirus 2019-nCoV is 1-14 days, and is mostly 3-7 days. More importantly, the latent period is also infectious, and asymptomatic infectors can also become a source of infection. Therefore, a novel and high-sensitivity detection method is urgently needed, and a solution is provided for epidemic situation prevention and control.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a novel coronavirus 2019-nCoV nucleic acid kit and a virus nucleic acid collection method.
The object of the present invention is achieved by the following technical means. A kit for rapidly and accurately detecting novel coronavirus 2019-nCoV nucleic acid by combining a Digital PCR (polymerase chain reaction) probe method is characterized in that a specific primer and a fluorescent probe are designed for detecting the novel coronavirus 2019-nCoV nucleic acid in a sample according to the novel coronavirus 2019-nCoV ORF1ab and an N gene as amplification target regions;
Target 1(ORF1ab):
forward primer (F): CCCTGTGGGTTTTACACTTAA
Reverse primer (R): ACGATTGTGCATCAGCTGA
Fluorescent probe (P): 5'-HEX-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'
Target 2(N gene):
forward primer (F): GGGGAACTTCTCCTGCTAGAAT
Reverse primer (R): CAGACATTTTGCTCTCAAGCTG
Fluorescent probe (P): 5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ 1-3'.
The invention provides a virus nucleic acid collection method, which comprises the steps of combining prepared Super premix reaction liquid A with Super premix reaction liquid B and C, carrying out Digital PCR absolute quantification on 2019-nCoV nucleic acid RNA by adopting a one-step method, carrying out Digital PCR detection on diluted positive virus sample RNA, and carrying out annealing treatment on a sample virus RNA at the temperature of 60.0 ℃ by taking the final concentration of a primer of 900nM and the final concentration of a probe of 300nM as optimal reaction concentrations and taking an ORF1ab probe and a probe of an N gene; detecting 2019-nCoV by two groups of specific probes of ORF1ab and N gene; the test result is judged according to the following steps: both HEX and FAM fluorescence channels of the sample to be detected are positive, and can be judged as 2019n-CoV virus nucleic acid positive, the positive quality control channel is positive, and the negative quality control channel is No call; on the contrary, the sample to be tested is judged to be negative.
The invention has the beneficial effects that: through a novel virus nucleic acid acquisition method and a Digital PCR probe method, the nucleic acid positive detection rate of the novel coronavirus 2019-nCoV infection is improved, the process specification and the standard are ensured, the influence of manual and non-standard operation on the detection result is reduced, the reliability of the result is ensured, and meanwhile, the false negative result caused by the combination of the sampling of sputum and a throat swab and the detection of a real-time fluorescence PCR method is avoided.
Drawings
FIG. 1 is a schematic representation of the HEX fluorescence channel copy number of the present invention.
FIG. 2 is a schematic representation of FAM fluorescence channel copy number of the present invention.
Detailed Description
The invention will be described in detail with reference to the following figures and examples:
1. design of specific primers and probes: according to the invention, a specific primer and a fluorescent probe (a sequence published by the Chinese disease prevention and control center) are designed according to the fact that the novel coronavirus 2019-nCoV ORF1ab (open reading frame 1ab) and the N gene (nucleocapsid protein) are used as amplification target regions, the specific primer and the fluorescent probe are used for detecting the novel coronavirus 2019-nCoV nucleic acid in a sample, and the detection specificity of the invention is ensured
Target 1(ORF1ab):
Forward primer (F): CCCTGTGGGTTTTACACTTAA
Reverse primer (R): ACGATTGTGCATCAGCTGA
Fluorescent probe (P): 5'-HEX-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'
Target 2(N gene):
forward primer (F): GGGGAACTTCTCCTGCTAGAAT
Reverse primer (R): CAGACATTTTGCTCTCAAGCTG
Fluorescent probe (P): 5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ1-3'
According to the sequence published by the Chinese disease prevention and control center, the invention modifies different fluorescence on the probe to realize single-tube one-step detection. The above primers and probes are all inBaori doctor Tech technology (Beijing) Co Ltd (takara China)Synthesized and purified by HPLC.
2. The novel virus nucleic acid collection method comprises the following steps: gauze mask collection (containing active carbon, polypropylene and other materials)
Based on the characteristics of the 2019-nCoV novel coronavirus, the special mask is prepared to collect viruses (such as cough and the like) discharged from respiratory tracts of patients, and then the mask is rinsed by using a virus dissolving solution and then enriched, so that the abundance of virus load can be improved, and the detection rate is greatly improved.
3. Based on the experiment principle of Digital PCR, the method mainly comprises a Super premix reaction solution and a quality control product:
super premix reaction liquid A: (including hot start Taq enzyme, RT enzyme, DTT, specific primer and probe, buffer solution, etc.)
Super premix reaction solution B (oil produced by probe liquid drop)
Super premix reaction solution C (drop reader oil)
Quality control (negative quality control in Table 1 and positive quality control in Table 2)
TABLE 1 negative reference compositions
| Numbering | Components | Concentration of |
| Negative 1 | Adenoviral vectors | 1000copies/μl |
| Negative 2 | Influenza A virus | 1000copies/μl |
| Negative 3 | Respiratory syncytial virus | 1000copies/μl |
| Negative 4 | MERS coronavirus pseudovirus | 1000copies/μl |
| Negative 5 | Pseudovirus of SARS coronavirus | 1000copies/μl |
| Negative 6 | Parainfluenza virus | 1000copies/μl |
| Negative 7 | Coronavirus HKU1 | 1000copies/μl |
| Negative 8 | Coronavirus NL63 | 1000copies/μl |
| Negative 9 | Coronavirus 229E | 1000copies/μl |
| Negative 10 | Coronavirus OC43 | 1000copies/μl |
TABLE 2 Positive reference Components
| Numbering | Components | Concentration of |
| Positive 1 | 2019-nCoV pseudovirus | 1000copies/μl |
| Positive 2 | 2019-nCoV pseudovirus | 100copies/μl |
| Positive 3 | 2019-nCoV pseudovirus | 10copies/μl |
And combining the prepared Super premix reaction liquid A with the Super premix reaction liquids B and C to perform Digital PCR detection. The group is positive and negative quality control products and samples to be detected, and a proper instrument is selected and carried out according to an operation instruction.
And (3) judging a detection result: the HEX and FAM fluorescence channels of the sample to be detected are positive, the sample to be detected 2019n-CoV virus nucleic acid can be judged to be positive, the positive quality control channel is positive, and the negative quality control channel is No call. On the contrary, the sample to be tested is judged to be negative.
The invention adopts a one-step method to carry out Digital PCR absolute quantification on 2019-nCoV nucleic acid RNA, and detects 2019-nCoV through two groups of specific probes of ORF1ab and N gene. The invention also optimizes each element, and has the advantages of simpler operation, higher sensitivity, less pollution and the like.
Compared with a fluorescence PCR method, the invention detects 2019-nCoV positive samples and has the advantages of high sensitivity, low detection limit and the like.
After the laboratory virus is enriched, the RNA concentration of the positive sample is 10-1～10-7And (4) diluting in multiple proportion, and performing RT-fluorescence PCR method and RT-Digital PCR method for quantification. The quantitative results of RT-Digital PCR method are shown in Table 3, and the copy numbers of HEX (ORF1ab) fluorescence channel are respectively: 6840. 670, 69, 6.8, 5.4, 4.2, 1.6copies/μ l; copy numbers of FAM (N gene) fluorescent channels were: 6350. 640, 63, 6.6, 5.1, 4.3, 1.8copies/μ l. The CT value of the RT-fluorescence PCR amplification curve is shown in Table 4, when the dilution is 10-7, the RT-fluorescence PCR and the negative control have no obvious amplification, no CT value is displayed, therefore, the sensitivity of the RT-fluorescence PCR method is that the dilution is 10-6Concentration of positive sample, Ct value of HEX (ORF1ab) 34.77; the Ct value of FAM (N gene) was 35.45 (less than 37). And the RT-Digital PCR method is carried out under the condition that the microdroplet generation is normal and the Poisson distribution is met and the dilution degree is 10-7The copy number concentrations of the positive samples were found to be 3.6 and 3.1 copies/. mu.L, respectively. The result shows that the lowest detection limit of the RT-Digital PCR method is as low as the number of single-bit copies, and the sensitivity of the RT-Digital PCR method is higher by one order of magnitude than that of the RT-fluorescent PCR method.
TABLE 3RT-Digital PCR method results
| Sample dilution | HEX (ORF1ab) copy number | FAM (N Gene) |
| 10-1 | 6480copies/μl | 6350copies/ |
| 10-2 | 670copies/μl | 640copies/ |
| 10-3 | 69copies/μl | 63copies/ |
| 10-4 | 6.8copies/μl | 6.6copies/ |
| 10-5 | 5.4copies/μl | 5.1copies/ |
| 10-6 | 4.2copies/μl | 4.3copies/ |
| 10-7 | 1.6copies/μl | 1.8copies/μl |
TABLE 4 RT-fluorescent PCR method results
| Sample dilution | HEX (ORF1ab) CT value | FAM (N Gene) |
| 10-1 | 20.14 | 20.77 |
| 10-2 | 23.16 | 23.76 |
| 10-3 | 26.42 | 26.89 |
| 10-4 | 28.43 | 29.13 |
| 10-5 | 30.26 | 31.39 |
| 10-6 | 32.51 | 33.24 |
| 10-7 | Unknow | Unknow |
According to the invention, by detecting the 2019-nCoV positive sample, enteroviruses and adenoviruses, as shown in the table 5, the HEX copy number of the 2019-nCoV positive sample is 488 copies/. mu.l; FAM fluorescence channel copy number 465 copies/. mu.l. The detection results of the enterovirus and the adenovirus are both No Call, which shows that the invention has high specificity.
TABLE 5 2019-nCoV Positive samples, Enterovirus and adenovirus results of the invention
| RT-Digital PCR method | 2019-nCoV positive sample | Enterovirus (EV) | Adenoviral vectors |
| HEX (ORF1ab) copy number | 488copies/μl | No Call | No Call |
| FAM (N Gene) copy number | 465copies/μl | No Call | No Call |
The invention detects the same 2019-nCoV positive sample at different times, and combines the standard deviation and the relative standard deviation of the experimental result, as shown in tables 6 and 7. The results of the test sections of the invention are shown to have good reproducibility both between groups and within groups.
Table 6: HEX fluorescence channel number
| Degree of dilution | Day one | The fifth day | The tenth day | Standard Deviation (SD) | Mean value of | Relative standard deviation (RSD%) |
| 10-1 | 8763 | 8923 | 9012 | 103.02 | 8899.33 | 1.16 |
| 10-2 | 865 | 833 | 912 | 32.44 | 870.00 | 3.73 |
| 10-3 | 76 | 56 | 67 | 8.18 | 66.33 | 12.33 |
| 10-4 | 8.5 | 7.6 | 8.3 | 0.39 | 8.13 | 4.74 |
| 10-5 | 6.5 | 6.2 | 5.7 | 0.33 | 6.13 | 5.38 |
| 10-6 | 3.4 | 3.1 | 2.9 | 0.21 | 3.13 | 6.56 |
Table 7: FAM fluorescence channel number
| Degree of dilution | Day one | The fifth day | The tenth day | Standard Deviation (SD) | Mean value of | Relative standard deviation (RSD%) |
| 10-1 | 9256 | 9873 | 8903 | 400.86 | 9344.00 | 4.29 |
| 10-2 | 945 | 965 | 876 | 38.13 | 928.67 | 4.11 |
| 10-3 | 89 | 98 | 92 | 3.74 | 93.00 | 4.02 |
| 10-4 | 9.1 | 8.9 | 9.7 | 0.34 | 9.23 | 3.68 |
| 10-5 | 7.3 | 6.7 | 7.8 | 0.45 | 7.27 | 6.19 |
| 10-6 | 4.3 | 4.7 | 3.8 | 0.37 | 4.27 | 8.63 |
Examples
The specific experimental steps of the invention are as follows:
extracting virus RNA from positive specimen (TaKaRa Mini BEST Viral RNA/DNA extraction kit)
1. Splitting virus in stock solution: 200. mu.l of the virus stock solution was added with 200. mu.l of Buffer VGB and 20. mu.l of protease K, and mixed well in a 56 ℃ water bath for 10 minutes. Add 200. mu.l absolute ethanol to the lysate, suck well and mix well.
2. The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12,000rpm for 2 minutes, and the filtrate was discarded.
3. Mu.l of Buffer RWA was added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.
4. Mu.l of Buffer RWB was added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.
5. And repeating the operation step 4.
6. Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes.
7. Spin Column was mounted on a new 1.5ml RNase free collection tube, and 30-50. mu.l of RNase free dH2O was added to the center of the Spin Column membrane, and allowed to stand at room temperature for 5 minutes.
The RNA was eluted by centrifugation at 8.12,000 rpm for 2 minutes.
And 9, detecting the RNA concentration. RT-Digital PCR Experimental procedure (QX 200 PCR instrument from Bole Co.)
Droplet generation:
preparing 20 mul of probe reaction system:
| reaction system | Volume (μ l) |
| Super premix reaction solution A | 18 |
| |
2 |
Transferring the above-mentioned solution into a microdroplet-generating cartridge, adding 70. mu.l of Super premix reaction solution B of the present invention, and placing the cartridge in a microdroplet-generating apparatus AutoDGTMAbove, the droplet starts to be generated.
PCR amplification
1. The automated droplet generator removes a microtiter plate containing droplets. Seal Plate with PX1 PCR Plate Sealer
2. Performing thermocycling amplification
Droplet reading
After amplification, the 96-well plate is placed on a QX200 reader, the Super premix reaction solution C of the invention is added, and QuantSoft software is opened for droplet reading. Negative quality control substances are added into the negative control group in each experiment to replace reaction templates.
Since the optimal reaction concentrations and annealing temperatures of the primers and probes are critical to the specificity and sensitivity of the invention, the optimization of the concentration of the Digital PCR probes and the optimization of the annealing temperature of the Digital PCR are performed in the process of the invention.
Optimization of probe concentration:
according to the experimental steps, the diluted positive virus sample RNA is subjected to Digital PCR detection, and the optimal condition of the probe concentration is explored.
In the experiment, 5 different probe final concentrations of 900nM primer and 500, 400, 300, 200 and 100nM from left to right are selected respectively. As a result, as shown in Table 8, from the viewpoint of the fluorescence signal intensity, the higher the probe concentration, the stronger the copy number signal. When the concentration of ORF1ab and N gene probe is 500nM or 400nM, the reaction has too high fluorescence signal intensity and positive amplification inhibition occurs. When the concentration of the probe is 200 or 100nM, the intensity of the fluorescence signal of the reaction is weaker; thus, it was shown from the experimental results that the sample viral RNA was at the optimal response concentration with both the primer final concentration of 900nM and the probe final concentration of 300 nM.
TABLE 8 Probe concentration gradient results
| Concentration of Probe | HEX (ORF1ab) copy number | FAM (N Gene) copy number |
| 500nM | 435copies/μl | 445copies/μl |
| 400nM | 523copies/μl | 519copies/μl |
| 300nM | 786copies/μl | 762copies/μl |
| 200nM | 598copies/μl | 593copies/μl |
| 100nM | 387copies/μl | 391copies/μl |
Optimization of probe annealing temperature:
and carrying out Digital PCR detection on the diluted positive virus sample RNA according to the optimized probe concentration, and exploring the optimal condition of the probe annealing temperature.
As shown in Table 9, the ORF1ab probe was used to perform RT-Digital PCR on samples at 65.0 ℃, 63.8 ℃, 62.8 ℃, 61.5 ℃, 60.0 ℃, 57.9 ℃, 56.8 ℃ and 55.0 ℃ at different annealing temperatures. The number of droplets is plotted on the abscissa and the intensity of the fluorescence signal is plotted on the ordinate, and the number of droplets and the number of copies are read directly on Quantasoft software, and the number of copies of the samples detected in the experiment is 344, 356, 378, 398, 469, 412, 388 and 365 copies/. mu.l, respectively. The probes for the N gene had copy numbers of 354, 376, 373, 412, 508, 455, 372 and 324 copies/. mu.l, respectively, under the same annealing temperature gradient. The results show that the copy number of the detected sample is the highest when the temperature of the ORF1ab probe and the N gene is 60.0 ℃, so that the optimal annealing temperature of the reaction is 60.0 ℃, and the reliability of the invention is greatly improved.
TABLE 9 Probe annealing temperature gradient results
The linear analysis and the determination of the lowest detection limit of the invention:
after extracting the RNA of the nucleic acid virus, the RNA is quantified by a NanoDrop Lite ultraviolet spectrophotometer, the concentration is 3.56 × 106 copies/mu l, the RNA is used as a standard substance to be diluted by 10 times, the concentration is S0-S7:3.56 × 106, 3.56 × 105, 3.56 × 104, 3560, 356, 35.6, 3.5 copies/mu l.RT-Digital PCR detection is carried out, the copy numbers of the HEX fluorescence channel and the FAM fluorescence channel are shown in table 10, LOG10 with the copy number of the standard substance is used as a vertical coordinate, the linear equation of the HEX fluorescence channel for RT-Digital PCR detection is y 1.0519x-0.4262, the correlation coefficient R2 is 0.9987, the linear equation of the FAM fluorescence channel for detection is y 1.0205x-0.3296, the correlation coefficient R2 is 0.9989, the linear relation is good, the invention is shown in a figure, the invention, the results can be determined by the quantitative result that the virus is detected by the linear equation of y-1.0205 x fluorescence channel, and the PCR detection is determined by the linear equation of the PCR method of the virus is the range of the virus of the NanoDrop V-9.
| S0 | S1 | S2 | S3 | S4 | S5 | S6 | S7 |
| Standard substance (copies/ul) | 3.56X106 | 3.56X105 | 3.56X104 | 3560 | 356 | 35.6 | 3.56 |
| HEX(copies/ul) | No call | 214563 | 24567 | 2214 | 212 | 18 | 1.13 |
| FAM(copies/ul) | No call | 196534 | 23234 | 1787 | 198 | 22 | 1.43 |
TABLE 10 Linear analysis and minimization of Standard
And (3) clinical specimen verification:
1. comparison of detection methods
The built Digital PCR system is utilized to carry out 2019-nCoV virus nucleic acid detection on clinical specimens. The same patient collects three times of throat swab specimens every 2 days, extracts viral RNA, and then respectively performs RT-fluorescence PCR method and RT-Digital PCR method to quantify viral nucleic acid. As shown in Table 11, the results of the two previous detections by RT-fluorescent PCR method showed no fluorescent signal; and when the sample collected for the third time is detected, HEX and FAM both have fluorescence amplification signals, the CT values are 33.56 and 32.22, and the sample virus nucleic acid is positive. According to the experimental steps of the invention, the detection result of the RT-Digital PCR method shows that the 3-time collected samples are all positive for virus nucleic acid, but the copy numbers of the viruses in the first two times are lower, and the HEX (ORF1ab) fluorescence channels are respectively 1.7 copies/mu l and 2.1 copies/mu l as shown in Table 12; the FAM (N gene) fluorescence channels were 1.9 copies/. mu.l and 2.3 copies/. mu.l, respectively. HEX (ORF1ab) in the third sample was 14.4 copies/. mu.l; FAM (N gene) was 13.2 copies/. mu.l. The invention has higher sensitivity and is important for early screening of epidemic situations.
TABLE 11 clinical specimen fluorescence PCR method test results
| Specimen collection | For the first time | For the second time | The third time |
| HEX (ORF1ab) CT value | Unknow | Unknow | 33.56 |
| FAM (N Gene) CT value | Unknow | Unknow | 32.22 |
TABLE 12 clinical specimen fluorescence PCR method detection results
| Specimen collection | For the first time | For the second time | The third time |
| HEX (ORF1ab) copy number | 1.7copies/μl | 4.1copies/μl | 14.4copies/μl |
| FAM (N Gene) copy number | 1.9copies/μl | 4.3copies/μl | 13.2copies/μl |
2. Comparison of specimen collections
The same patient in clinic respectively adopts a throat swab, sputum and the mask virus enrichment method. After the special mask is worn for 2 hours, virus RNA is rinsed and enriched, and then an RT-Digital PCR method is carried out to quantify the virus nucleic acid. As shown in the results of Table 13, the mask specimen enrichment method provided by the invention has the advantages that the copy numbers of the HEX (ORF1ab) fluorescence channel and the FAM (N gene) fluorescence channel are far greater than those of the throat swab and sputum specimen, so that the virus load abundance is greatly improved, and the 2019-nCoV virus nucleic acid detection sensitivity of the invention, namely the clinical specimen sputum and the mask enrichment method provided by the invention, are improved
| Specimen collection method | Throat swab | Sputum | Enrichment method for mask |
| HEX (ORF1ab) copy number | 3.5copies/μl | 5.8copies/μl | 25.4copies/μl |
| FAM (N Gene) copy number | 3.2copies/μl | 6.1copies/μl | 25.3copies/μl |
Through multiple experimental optimization, the method for detecting the nucleic acid absolute quantification of the 2019-nCoV virus by the one-step Digital PCR method is established on the basis of the mask specimen enrichment method, reduces the possible pollution in the two-step method and the fluorescence quantitative PCR method, and has the advantages of strong specificity, high sensitivity, low detection limit and the like. Has important significance for the detection, prevention and control of epidemic situations and the like.
It should be understood that equivalent substitutions and changes to the technical solution and the inventive concept of the present invention should be made by those skilled in the art to the protection scope of the appended claims.
Claims (2)
1. A novel coronavirus 2019-nCoV nucleic acid kit, which is characterized in that: according to the new coronavirus 2019-nCoV ORF1ab and the N gene as amplification target regions, designing a specific primer and a fluorescent probe for detecting the new coronavirus 2019-nCoV nucleic acid in a sample;
Target 1(ORF1ab):
forward primer (F): CCCTGTGGGTTTTACACTTAA
Reverse primer (R): ACGATTGTGCATCAGCTGA
Fluorescent probe (P): 5'-HEX-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'
Target 2(N gene):
forward primer (F): GGGGAACTTCTCCTGCTAGAAT
Reverse primer (R): CAGACATTTTGCTCTCAAGCTG
Fluorescent probe (P): 5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ 1-3'.
2. A method for collecting viral nucleic acids using the novel coronavirus 2019-nCoV nucleic acid kit of claim 1, wherein: performing Digital PCR absolute quantification on 2019-nCoV nucleic acid RNA by adopting a one-step method, performing Digital PCR detection on diluted positive virus sample RNA, and performing annealing treatment on the ORF1ab probe and the probe of the N gene at the temperature of 60.0 ℃ by taking 900nM primer final concentration and 300nM probe final concentration of sample virus RNA as optimal reaction concentrations; detecting 2019-nCoV by two groups of specific probes of ORF1ab and N gene; the test result is judged according to the following steps: both HEX and FAM fluorescence channels of the sample to be detected are positive, and can be judged as 2019n-CoV virus nucleic acid positive, the positive quality control channel is positive, and the negative quality control channel is No call; on the contrary, the sample to be tested is judged to be negative.
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