CN113337642A - RT-PCR kit for detecting novel coronavirus - Google Patents
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Abstract
The invention relates to the technical field of virus detection, in particular to an RT-PCR kit for detecting novel coronavirus. According to the invention, the novel coronavirus RT-PCR kit is prepared by optimizing the nucleic acid release condition, the positive and negative control fluorescent RT-PCR conditions and the fluorescent RT-PCR reaction condition of the nucleic acid sample, and compared with the existing detection kit, the detection time is obviously shortened, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to an RT-PCR kit for detecting novel coronavirus.
Background
The nucleic acid detection of the new coronavirus is an effective means for rapidly screening various populations such as clinical patients and suspected patients with different degrees, and the result can be used as the most direct evidence for clinical diagnosis. According to the diagnostic protocol for novel coronavirus pneumonia in China (trial eighth edition), the aetiological evidence for confirmed diagnosis of cases includes real-time fluorescence RT-PCR monitoring of novel coronavirus nucleic acid positivity and virus gene sequencing, which are highly homologous with the known novel coronavirus. The current real-time fluorescence RT-PCR detection has the characteristics of high sensitivity and strong specificity, is a nucleic acid detection method with wider application, and is particularly suitable for large-scale screening.
With the development of a large amount of detection work, the positive detection rate of the nucleic acid detection kit is only 30-50%, multiple suspected patients show positive results after 2-3 times of nucleic acid detection, and even some patients have obvious symptoms, but the nucleic acid detection is still negative for many times; but also the patient who is discharged has the phenomenon of recovering yang. The reasons include sampling time, sampling method, sample transportation and other links, and different nucleic acid detection test cassettes and extraction methods are also important factors for false negative results. Therefore, there is a need to develop a new kit for detecting coronas with higher positive detection rate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the RT-PCR kit for detecting the novel coronavirus, and the kit provided by the invention greatly shortens the time for detecting the 2019-nCoV virus and improves the detection efficiency and the detection accuracy.
In order to achieve the purpose, the invention adopts the following scheme:
the kit comprises 2019-nCoV RT-PCR detection reaction liquid, wherein the 2019-nCoV RT-PCR detection reaction liquid comprises specific primers of an ORF1ab gene, an N gene and an RNase P gene; the nucleotide sequence of the upstream primer of the ORF1ab gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the N gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the RNase P gene is shown as SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.
Further, the 2019-nCoV RT-PCR detection reaction solution also comprises probes of an ORF1ab gene, an N gene and an RNase P gene, wherein the probe nucleotide sequence of the ORF1ab gene is shown as SEQ ID NO. 7, the probe nucleotide sequence of the N gene is shown as SEQ ID NO. 8, and the probe nucleotide sequence of the RNase P gene is shown as SEQ ID NO. 9.
Further, the ORF1ab gene probe is modified into FAM at the 5 'part and BHQ1 at the 3' part; the 5 'of the N gene probe is modified into HEX, and the 3' of the N gene probe is modified into BHQ 1; the 5 'modification of the RNase P gene probe is Cy5, and the 3' modification is BHQ 3.
Further, the 2019-nCoV RT-PCR detection reaction solution also comprises Buffer and RNase Free dH2O, and the kit also comprises RT-PCR enzyme, a 2019-nCoV positive control, a 2019-nCoV negative control, a 2019-nCoV sample nucleic acid releasing agent A solution and a 2019-nCoV sample nucleic acid releasing agent B solution.
Further, the RT-PCR enzyme is Taq enzyme and reverse transcriptase.
Further, the 2019-nCoV positive control substance has a concentration of 1062019-nCoV-CDC positive plasmid with copies/mL, the 2019-nCoV negative control product is RNase Free dH2O, the 2019-nCoV sample nucleic acid releaser A solution is prepared from sodium hydroxide, triton X-100, Tris-HCl, ethylphenyl polyethylene glycol, Tween 20, sodium chloride, potassium chloride, proteinase K and lithium dodecyl sulfate, the 2019-nCoV sample nucleic acid releaser B solution is prepared from triton X-100, Tris-HCl,tween 20, ethylphenyl polyethylene glycol, sodium chloride and potassium chloride.
Furthermore, the molar concentration of sodium hydroxide in the solution A of the nucleic acid releasing agent is 25mM to 100mM, the volume percentage of triton X-100 is 0.1 percent to 3 percent, the molar concentration of Tris-HCl is 0.5mM to 10mM, the volume percentage of ethyl phenyl polyethylene glycol is 0.1 percent to 3 percent, the volume percentage of Tween 20 is 0.1 percent to 3 percent, the mass concentration of lithium dodecyl sulfate is 0.2mg/mL to 20mg/mL, the mass concentration of protease K is 0.1mg/mL to 5mg/mL, the molar concentration of sodium chloride is 20mM to 200mM, and the molar concentration of potassium chloride is 20mM to 200 mM.
Furthermore, the volume percentage of the triton X-100 in the solution B of the nucleic acid releasing agent is 0.1-3 percent, the molar concentration of Tris-HCl is 0.5-10 mM, the volume percentage of Tween 20 is 0.1-3 percent, the volume percentage of ethyl phenyl polyethylene glycol is 0.1-3 percent, the molar concentration of sodium chloride is 20-200 mM, and the molar concentration of potassium chloride is 20-200 mM.
Further, the reaction system of the kit is as follows:
further, the reaction conditions of the kit are as follows:
reverse transcription: 5min at 42 ℃ for 1 cycle;
pre-denaturation: 10sec at 95 ℃ for 1 cycle;
and (3) PCR: 95 ℃ for 5 sec: 20sec at 60.8 ℃ for 40 cycles, and fluorescence was collected at the end of each 60.8 ℃.
Furthermore, the sample detected by the kit is a virus sample inactivated at 56 ℃ for 30 min.
Specifically, for the nucleic acid detection of 2019-nCoV, a sample is required to be collected, and the conventional sample types comprise pharyngeal swabs, nasal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid and the like. 2019-nCoV is a single-stranded RNA virus, and RNA is easy to degrade. Therefore, it is necessary to use an RNase-free swab and an RNase-free storage tube when collecting a sample. In order to ensure the detection accuracy and eliminate false negative results, a human-derived reference gene can be added into a PCR reaction system, the reverse transcription and amplification reaction is monitored in the whole process, and the quality guarantee of the collected sample is realized.
Further, 1) the virus-inactivated sample and the 2019-nCoV sample nucleic acid releasing agent A solution were left at 65 ℃ for 5min in an RNase free collection tube;
2) and adding the nucleic acid releasing agent B, oscillating for 5-10 sec, and standing at 25 ℃ for 10min to obtain a sample mixed solution to be detected.
The principle is as follows: based on newly published gene sequence information of the novel coronavirus 2019-nCoV, the invention designs a double-target gene and a Taqman fluorescent probe aiming at a specific gene sequence of the novel coronavirus 2019-nCoV, and realizes qualitative detection of RNA of the novel coronavirus 2019-nCoV in samples such as nasopharyngeal swabs, sputum, lower respiratory tract secretion, blood, excrement and the like.
Compared with the prior art, the invention has the following beneficial effects:
by optimizing the nucleic acid release condition, the positive and negative control fluorescence RT-PCR condition and the fluorescence RT-PCR reaction condition of the nucleic acid sample, the detection rate of the medium/strong positive sample of the prepared RT-PCR kit for detecting the novel coronavirus is 100%, the detection rate of the negative sample is 100%, the minimum detection limit is determined to be 960copies/mL, the detection rate of the minimum detection limit positive is more than or equal to 95%, the coefficient of variation CV of Ct precision is less than or equal to 5%, compared with the existing detection kit, the detection time is obviously shortened (15 min for nucleic acid extraction and 45min for reverse transcription and amplification reaction), the detection efficiency is improved, and the kit can play an important role in the new corona epidemic situation.
Drawings
FIG. 1 is a graph showing the fluorescent signal acquisition of RNase P-ROX, a probe of example 1 of the present invention.
FIG. 2 is a graph showing the fluorescent signal collection of the probe RNase P-Cy5 according to example 1 of the present invention.
FIG. 3 is a graph showing the fluorescent signal acquisition of probe R1-ROX in example 1 of the present invention.
FIG. 4 is a graph showing the collection of fluorescence signals of the probe R1-Cy5 according to example 1 of the present invention.
FIG. 5 is a graph comparing the intensity of fluorescence signals of Cy5 and ROX in example 1 of the present invention.
FIG. 6 shows a graph of the results of comparison of 1X 10 pairs under the condition 1 in example 2 of the present invention2～1×109copies/mL positive plasmid amplification plot and line plot.
FIG. 7 shows the results of comparison of 1X 10 pairs under the condition 2 in example 2 of the present invention2～1×109copies/mL positive plasmid amplification plot and line plot.
FIG. 8 shows the results of comparison of 1X 10 pairs under the condition 3 in example 2 of the present invention2～1×109copies/mL positive plasmid amplification plot and line plot.
FIG. 9 shows different processing time length pairs of 1 × 10 in embodiment 3 of the present invention6And 1X 104copies/mL 2019-nCoV pseudovirus fluorescent RT-PCR FAM channel detection map.
FIG. 10 shows different processing time length pairs of 1 × 10 in embodiment 3 of the present invention6And 1X 104copies/mL 2019-nCoV pseudovirus fluorescent RT-PCR HEX channel detection map.
FIG. 11 is a line graph showing the setting threshold values of the negative control group in example 3 of the present invention.
FIG. 12 shows a pair 10 of example 3 of the present invention6Fluorescent RT-PCR FAM channel detection map of copies/mL 2019-nCoV pseudovirus concentration.
FIG. 13 shows a pair 10 of example 3 of the present invention6Fluorescent RT-PCR HEX channel detection map of copies/mL 2019-nCoV pseudovirus concentration.
FIG. 14 is a line graph of inactivation treated group in example 4 of the present invention.
FIG. 15 is a line graph of the uninhibited group of samples of example 4 of the present invention.
FIG. 16 is a fluorescent RT-PCR amplification chart of the negative control group in example 7 of the present invention.
FIG. 17 is the fluorescent RT-PCR amplification chart of SARS group in example 7 of the present invention.
FIG. 18 is a fluorescent RT-PCR amplification chart of MERS group in example 7 of the present invention.
FIG. 19 is a 2019-nCoV group fluorescence RT-PCR amplification chart of the invention in example 7.
FIG. 20 is the fluorescent RT-PCR amplification chart of SARS +2019-nCoV group in example 7 of the present invention.
FIG. 21 is a MERS +2019-nCoV group fluorescence RT-PCR amplification chart in example 7 of the present invention.
FIG. 22 is a fluorescent RT-PCR amplification chart of SARS + MERS +2019-nCoV group in example 7 of the present invention.
Detailed Description
The technical solution of the present invention is further explained with reference to the drawings and the embodiments.
Example 1 reference Gene primers and Probe channel selection
Two pairs of primers (RNase P and R1) and corresponding probes (ROX and Cy5) are designed according to the RNase P gene sequence for comparison and amplification efficiency, the nucleotide sequence of the upstream primer of R1 is shown as SEQ ID NO:10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:11, the nucleotide sequence modified by the probe CY5 is shown as SEQ ID NO:12, the nucleotide sequence modified by the ROX is shown as SEQ ID NO:13, and the nucleotide sequence of the ROX modified probe of the RNase P is shown as SEQ ID NO: 14. The results of each experimental group were observed from the baseline using whole blood RNA as a sample, and as can be seen from FIGS. 1 to 4, there were amplification curves for RNase P-ROX and RNase P-Cy5, while the results for R1-ROX and R1-Cy5 showed no amplification. The same primers RNase P-F and R are used for RNase P-ROX and RNase P-Cy5, and the same primers R1-Fand R are used for R1-ROX and R1-Cy 5; meanwhile, the primer and the probe are designed in a matching way, which shows that the amplification efficiency of the RNase P is higher than that of the R1 in the aspect of the primer (FIG. 5).
Example 2 Triplex fluorescent PCR annealing temperature optimization
And optimizing the temperature of the triple fluorescence PCR, and drawing a standard curve according to the selected annealing temperature. As shown in fig. 6-8, 3 different temperature conditions were set: at 1.95 deg.C for 1min for 1 cycle; 40 cycles of 95 ℃ for 5sec and 61.3 ℃ for 30 sec; at 2.95 deg.C for 1min for 1 cycle; 40 cycles of 95 ℃ for 15sec and 60.8 ℃ for 20 sec; at 3.95 deg.C for 1min for 1 cycle; 5sec at 95 ℃ and 20sec at 60.8 ℃ for 40 cycles. And finally, optimizing the PCR reaction program to obtain the following reaction program: reverse transcription 5min at 42 ℃, 10sec at 95 ℃ (for RNA samples), or 1min at 95 ℃ (only heavyGroup plasmids) for 1 cycle; the fluorescence signal was collected after the end of each 60.8 ℃ cycle, 40 cycles of 95 ℃ for 5sec and 60.8 ℃ for 30 sec. The detection channel is selected from FAM, HEX and Cy 5. Under these conditions, the plasmid was at 10-1～10-5ng/mu L is in linear relation and the correlation coefficient R of a standard curve2Both are more than or equal to 0.995, and the amplification efficiencies of ORF1ab and the N gene are 87.1% and 92.5%, respectively.
Example 3 optimization of nucleic acid Release Agents
Using different processing time length pairs of 1X 106And 1X 104Fluorescence RT-PCR detection is carried out on ORF1ab and N genes of copies/mL 2019-nCoV pseudovirus, FAM and HEX channels are selected, and after a threshold line is set according to a negative group, the Ct value statistical result of an experimental group is analyzed: 1. judged by the NEG group results, 10 in the detection6And 104Both copies/ml can be read as positive results; 2. the difference between the fluorescence intensity (figure 9-10) signals and Ct values corresponding to ORF1ab and N genes is found to be small by comparison of treatment (5-20 min) with the same concentration and different time durations, and the nucleic acid can be extracted within 5 min.
Setting different dosage ratios of pseudoviruses and nucleic acid releasing agents, wherein the ratio is 1, 50: 50: 50; ratio 2, 10: 50: 50; ratio 3, 5: 100: 50; ratio 4, 5: 50: 50; then, performing fluorescence RT-PCR detection on ORF1ab and N genes of the 2019-nCoV pseudovirus, selecting FAM and HEX channels, setting a threshold line according to a negative group, then obviously (shown in figures 11-13) having an obvious amplification curve, and counting the analysis result of the Ct value: changing the dosage ratio of the pseudovirus and the nucleic acid releaser to have obvious influence on the minimum detection limit of a reaction system, therefore, in the step of nucleic acid extraction, 50 mu L of pseudovirus, nucleic acid releaser A liquid and nucleic acid releaser B liquid are sampled, the 2019-nCoV sample nucleic acid releaser A liquid is prepared from sodium hydroxide, triton X-100, Tris-HCl, ethylphenylpolyethylene glycol, Tween 20, sodium chloride, potassium chloride, protease K and lithium dodecyl sulfate, and the 2019-nCoV sample nucleic acid releaser B liquid is prepared from triton X-100, Tris-HCl, Tween 20, ethylphenylpolyethylene glycol, sodium chloride and potassium chloride.
The molar concentration of sodium hydroxide in the solution A of the nucleic acid releasing agent is 25 mM-100 mM, the volume percentage of triton X-100 is 0.1% -3%, the molar concentration of Tris-HCl is 0.5 mM-10 mM, the volume percentage of ethyl phenyl polyethylene glycol is 0.1% -3%, the volume percentage of Tween 20 is 0.1% -3%, the mass concentration of lithium dodecyl sulfate is 0.2 mg/mL-20 mg/mL, the mass concentration of protease K is 0.1 mg/mL-5 mg/mL, the molar concentration of sodium chloride is 20 mM-200 mM, and the molar concentration of potassium chloride is 20 mM-200 mM.
The volume percentage of the triton X-100 in the solution B of the nucleic acid releasing agent is 0.1-3 percent, the molar concentration of Tris-HCl is 0.5-10 mM, the volume percentage of Tween 20 is 0.1-3 percent, the volume percentage of ethyl phenyl polyethylene glycol is 0.1-3 percent, the molar concentration of sodium chloride is 20-200 mM, and the molar concentration of potassium chloride is 20-200 mM.
Example 4 Effect of Virus inactivation treatment on the assay
2019-nCoV belongs to a virulent infectious disease, so that the virus is selected to be subjected to fire extinguishing treatment for detection. The concentration range is 2.0 multiplied by 106～3.2×103The copies/mL 2019-nCoV viruses were divided into the inactivated treated group and the non-inactivated treated group at 56 ℃ for 30min, as shown in FIGS. 13 to 14: experimental results show that the linear trend of an inactivated group is similar to that of an inactivated group in pseudoviruses with the same gradient dilution concentration, and the Ct values are counted to have no obvious difference, so that the nucleic acid releasing agent can still effectively extract and protect virus RNA after the inactivation treatment of the 2019-nCoV pseudoviruses at 56 ℃ for 30min, and the amplification effect of primers and probes on target gene segments is excellent.
Example 5 lowest detection Limit
After virus inactivation, the concentration was 3.0X 106～9.6×102Samples of copies/mL were tested as shown in Table 1: the linear trends of the same concentration are similar, the Ct values are similar, and the lowest detection limit can reach 6.4 multiplied by 102Although the Ct values of FAM and HEX channels of the copies/mL are close to 40, false negative experiment results are easily caused, and the low-concentration Ct value is excessively changed possibly because the nucleic acid releasing agent in sample treatment is related to uneven sample treatment and causes too low template concentration, so that the lowest detection limit is determined to be 9.6 multiplied by 102copies/mL。
TABLE 1 statistical Ct values for linear experiments
Example 6 precision
For the lowest detection limit of 9.6 × 102The samples/mL are used for daytime precision experiments, and N is more than or equal to 20 each time. FAM and HEX channel Ct were used as references (Table 2). 93 samples were tested in a total of 4 tests, including 87 double positive samples (93.55%), 4 single FAM positive samples (4.30%), and 2 single HEX positive samples (2.15%), wherein the FAM channel coefficient of variation CVs are 2.66%, 2.23%, 1.88%, and 2.29%, and the HEX channel coefficient of variation CVs are 2.09%, 1.59%, 2.25%, and 2.38%, respectively, for example, for the double positive samples. Prove that the kit developed by the subject can detect 9.6 multiplied by 102copies/mL, and the precision meets the requirement.
TABLE 2 Ct value statistics for precision experiments
Example 7 specificity test
Extracting ORF1ab and N gene of SARS, MERS and 2019-nCoV, and setting negative control group, SARS group, MERS group, 2019-nCoV group, SARS +2019-nCoV group, MERS +2019-nCoV group and SARS + MERS +2019-nCoV group to perform fluorescence RT-PCR detection. The results are shown in FIGS. 16-22 and Table 3: the same concentration of the three pseudoviruses (1.0X 10)6copies/mL), ORF1ab and N gene can be judged to be positive only in the 2019-nCoV group, although the SARS group and MERS group have the signal value of N gene at the same time, the Ct value is close to that of the negative group, and non-specific amplification is possible; 2019-nCoV with the same concentration is added into SARS and MERS groups, so that FAM and HEX values can be judged to be positive; the three pseudoviruses are mixed with the same concentration and then used as a template, and the Ct value can also be used for positive interpretation. And (3) the Ct value of the experimental group with the positive result accords with the fluorescent quantitative PCR principle, namely the higher the concentration of the target template is, the smaller the Ct value is. Proves that the specificity of the primer probe to the 2019-nCoV virus is good, even if SARS virus and MERS virus with extremely high genome homology exist in a sample, the primer probe still can be used for detecting the SARS virus and the MERS virusCan be detected specifically.
TABLE 3 statistical Ct values for specificity experiments
Example 8 detection method of novel coronavirus 2019-nCoV triple nucleic acid detection kit
The prepared kit is 24 persons, the main components are shown in table 4, the kit is stored at minus 20 +/-5 ℃ in a dark place, and the effective period of the product is 12 months. Repeated freezing and thawing of the RT-PCR detection reaction solution is not suitable for more than 5 times.
Inspection method
1. Reagent preparation (reagent preparation area)
1.1 taking out each component in the kit, standing at room temperature, and uniformly mixing for later use after the temperature of the components is balanced to the room temperature;
1.2 according to the number of samples to be detected, the number of positive controls and the number of negative controls, taking corresponding amount of components according to the proportion (19 mu L/person part of detection reaction liquid and 1 mu L/person part of RT-PCR enzyme), fully and uniformly mixing to obtain a PCR mixed solution, and subpackaging into 8-row PCR tubes every 20 mu L/person time after instantaneous centrifugation.
1.3 transferring the prepared reagent to a sample processing area for use.
2. Sample preparation (sample processing zone)
2.1 taking 50 μ L of sample which has finished virus inactivation and 50 μ L of 2019-nCoV sample nucleic acid releasing agent A liquid into 1.5mL of RNase free collection tube, and standing at 65 ℃ for 5 min;
2.2 adding 50 mul of the nucleic acid releasing agent B solution, oscillating for 5-10 sec, and standing for 10min at 25 ℃;
2.3 mu.L of the mixture was taken and put into a PCR amplification tube containing 20 mu of the LPCR mixture, and the fluorescent quantitative PCR was detected on a fluorescent PCR instrument.
PCR amplification (amplification and analysis region)
3.1 placing the PCR amplification tube into the sample groove of the amplification instrument, setting the sample number sequence and the negative/positive control hole according to the instrument operation instruction, and setting the sample name.
3.2PCR recommended cycle parameter settings:
reverse transcription: 5min at 42 ℃ for 1 cycle;
pre-denaturation: 10sec at 95 ℃ for 1 cycle;
and (3) PCR: 95 ℃ for 5 sec: 60.8 ℃ for 20 sec. For a total of 40 cycles. Fluorescence was collected at the end of each 60.8 ℃ and the detection channels were as follows:
FAM (this channel indicates the 2019-nCoV ORF1a/b gene detection);
HEX (this channel indicates 2019-nCoV N gene detection);
cy5 (the channel indicates the detection of the human reference gene RNase P).
And (4) positive judgment: the Ct reference value of the kit for detecting the target gene is determined to be 36 through reference value research.
The detection result shows that:
based on the instrument data, a reasonable baseline is set, and 3-15 cycles are recommended. The threshold was set to just exceed the highest point of the negative control amplification curve. The quality control conditions are shown in Table 5, 1. detecting a typical S-type amplification curve for FAM or HEX channels according to the detection result, reporting that the sample with the Ct less than or equal to 36 is 2019-nCoV virus positive; 2. for FAM samples in which a typical S-type amplification curve (N/A) is not detected in the HEX channel or Ct is greater than 36, the Cy5 channel has an amplification curve, and the Ct is less than or equal to 36, and the samples are reported as 2019-nCoV virus negative; 3. for the samples of which the FAM, HEX and Cy5 channels do not detect a typical S-shaped amplification curve (N/A) or Ct is larger than 36, the detection result of the sample is invalid, the reason should be searched and eliminated, the sample is resampled, and repeated tests are carried out or other factory kits are used for carrying out tests, wherein the sample has an excessively low cell content or has an interfering substance reaction.
TABLE 4 major ingredients
| Reagent | 24 persons per box |
| 2019-nCoV RT-PCR detection reaction solution | 520 μ l/tube |
| RT-PCR enzymes | 27 μ l/tube |
| 2019-nCoV |
50 μ l/tube |
| 2019-nCoV |
50 μ l/tube |
| 2019-nCoV sample nucleic acid releasing agent A liquid | 1.35 ml/piece |
| 2019-nCoV sample nucleic acid releasing agent B liquid | 1.35 ml/piece |
TABLE 5 quality control
Note: the above conditions need to be satisfied in the same experiment, and if the control does not meet the requirements, the experimental result is invalid.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
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Claims (10)
1. An RT-PCR kit for detecting novel coronavirus, which is characterized by comprising 2019-nCoV RT-PCR detection reaction liquid, wherein the 2019-nCoV RT-PCR detection reaction liquid comprises specific primers of ORF1ab gene, N gene and RNase P gene; the nucleotide sequence of the upstream primer of the ORF1ab gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the N gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the RNase P gene is shown as SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.
2. The RT-PCR kit for detecting the novel coronavirus according to claim 1, wherein the 2019-nCoV RT-PCR detection reaction solution further comprises probes of ORF1ab gene, N gene and RNase P gene, the probe nucleotide sequence of ORF1ab gene is shown as SEQ ID NO. 7, the probe nucleotide sequence of the N gene is shown as SEQ ID NO. 8, and the probe nucleotide sequence of the RNase P gene is shown as SEQ ID NO. 9.
3. The RT-PCR kit for detecting the novel coronavirus according to claim 2, wherein the ORF1ab gene probe is modified into FAM at the 5 'end and BHQ1 at the 3' end; the 5 'of the N gene probe is modified into HEX, and the 3' of the N gene probe is modified into BHQ 1; the 5 'modification of the RNase P gene probe is Cy5, and the 3' modification is BHQ 3.
4. The RT-PCR kit for detecting the novel coronavirus according to claim 1, wherein the reaction solution for detecting the 2019-nCoV RT-PCR further comprises Buffer and RNase Free dH2O, and the kit further comprises RT-PCR enzyme, a 2019-nCoV positive control, a 2019-nCoV negative control, a 2019-nCoV sample nucleic acid releasing agent A solution and a 2019-nCoV sample nucleic acid releasing agent B solution.
5. The RT-PCR kit for detecting novel coronavirus according to claim 4, wherein the RT-PCR enzyme is Taq enzyme and reverse transcriptase.
6. The RT-PCR kit for detecting the novel coronavirus according to claim 4, wherein the 2019-nCoV positive control is a 2019-nCoV-CDC positive plasmid with the concentration of 106copies/mL, the 2019-nCoV negative control is RNase Free dH2O, the 2019-nCoV sample nucleic acid releasing agent A liquid is prepared from sodium hydroxide, triton X-100, Tris-HCl, ethylphenyl polyethylene glycol, Tween 20, sodium chloride, potassium chloride, protease K and lithium dodecyl sulfate, and the 2019-nCoV sample nucleic acid releasing agent B liquid is prepared from triton X-100, Tris-HCl, Tween 20, ethylphenyl polyethylene glycol, sodium chloride and potassium chloride.
7. The RT-PCR kit for detecting the novel coronavirus according to claim 5, wherein the reaction system of the kit is as follows:
8. the RT-PCR kit for detecting the novel coronavirus according to any one of claims 1 to 7, wherein the reaction conditions of the kit are as follows:
reverse transcription: 5min at 42 ℃ for 1 cycle;
pre-denaturation: 10sec at 95 ℃ for 1 cycle;
and (3) PCR: 95 ℃ for 5 sec: 20sec at 60.8 ℃ for 40 cycles, and fluorescence was collected at the end of each 60.8 ℃.
9. The RT-PCR kit for detecting the novel coronavirus according to any one of claims 1-7, wherein the sample detected by the kit is a virus sample inactivated at 56 ℃ for 30 min.
10. The RT-PCR kit for detecting the novel coronavirus according to claim 9, wherein the method for extracting a sample to be detected of the kit is as follows:
1) placing virus inactivated sample and 2019-nCoV sample nucleic acid releasing agent A solution in RNase free collection tube at 65 deg.C for 5 min;
2) and adding the nucleic acid releasing agent B, oscillating for 5-10 sec, and standing at 25 ℃ for 10min to obtain a sample mixed solution to be detected.
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