CN112760415A

CN112760415A - Novel coronavirus, influenza A and B virus full-premix freeze-drying multiplex fluorescence PCR detection kit and detection method thereof

Info

Publication number: CN112760415A
Application number: CN202011571695.0A
Authority: CN
Inventors: 李然栋; 马清霞; 薄慧文
Original assignee: Qingdao Batfi Technology Development Co ltd
Current assignee: Qingdao Batfi Technology Development Co ltd
Priority date: 2020-12-27
Filing date: 2020-12-27
Publication date: 2021-05-07

Abstract

The invention discloses a multiple fluorescence PCR rapid detection kit for novel coronavirus, influenza A virus and influenza B virus, which comprises a freeze-dried solid RT-PCR Mix, a liquid redissolving Buffer, a freeze-dried solid positive control and a freeze-dried solid negative control, wherein the freeze-dried solid RT-PCR Mix contains a primer group corresponding to primer sequences of a SARS-CoV-2 specific gene ORF1ab, an influenza A M gene, an influenza B M gene and a human origin internal reference gene RNAse P. The detection method is simple and convenient to operate, has low requirement on the operation level of detection personnel, can detect 3 common respiratory pathogens at one time, greatly saves the detection time and the detection cost, realizes the rapid screening of a large batch of samples, only needs 40 minutes to 1 hour in the whole detection process, and has accurate and reliable results.

Description

Novel coronavirus, influenza A and B virus full-premix freeze-drying multiplex fluorescence PCR detection kit and detection method thereof

Technical Field

The invention belongs to the technical field of in-vitro diagnosis and biological detection, and particularly relates to a novel coronavirus, influenza A virus and influenza B virus full-premix freeze-drying multiplex fluorescence PCR detection kit and a detection method thereof.

Background

2019 coronavirus Disease (Corona Virus Disease 2019, COVID-19) is an acute respiratory infectious Disease caused by a novel coronavirus (New Corona Virus, SARS-CoV-2). SARS-CoV-2 belongs to a novel coronavirus of the genus beta, which is an RNA virus, which is enveloped, has a circular or elliptical particle, is usually polymorphic, has a diameter of 60-140nm, is the 7 th coronavirus which is known to infect humans at present, and the other 6 coronaviruses are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, respectively. The first 4 of these are more common in the population, less pathogenic, and generally cause only mild respiratory symptoms like the common cold, while the other 2 are the more pathogenic SARS coronavirus and MERS coronavirus.

Influenza virus (influenza) belongs to Orthomyxoviridae (Orthomyxoviridae), referred to as influenza virus for short, and includes human influenza virus and animal influenza virus, and the human influenza virus is classified into three types, i.e., A (influenza virus), B (influenza virus) and C (influenza virus) and is a pathogen of influenza (flu). The influenza A virus can naturally infect people and various animals, often suddenly occurs and spreads rapidly, and causes a worldwide pandemic for many times; influenza b viruses only infect humans in nature, generally in sporadic, outbreak or small circulation; influenza c virus often infects children, often sporadically, sporadically or outbreaks, but not epidemic. The surface antigen Hemagglutinin (HA) of influenza A virus is easy to mutate with Neuraminidase (NA), and it is known that HA HAs 16 subclasses (H1-H16) and NA HAs 9 subclasses (N1-N9), and random combination of them can form a plurality of subtypes without cross immunity among subtypes. This variable nature makes diagnosis and control of influenza very difficult.

In autumn and winter, the influenza is a time period with high incidence of influenza, and under the current situation of global epidemic of new coronary, the influenza and the new coronary pneumonia are very needed to be distinguished for targeted treatment, so that the cure rate is improved. However, the currently widely adopted method is that after a patient is sampled, new coronary pneumonia is firstly detected, and then each subtype influenza is detected, so that time is consumed, and the detection rate of viruses is influenced due to repeated freezing and thawing of the sample. Therefore, a method and a kit which have high sensitivity and strong specificity and can simultaneously detect the pathogens of 3 common respiratory diseases such as novel human coronavirus, influenza A virus, influenza B virus and the like are urgently needed in the market.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a novel triple fluorescence PCR rapid detection kit for coronavirus, influenza A virus and influenza B virus and a detection method thereof.

The invention provides a novel coronavirus, influenza A and B virus triple fluorescence PCR rapid detection kit, which comprises a freeze-dried solid RT-PCR Mix, a liquid redissolving Buffer, a freeze-dried solid positive control, a freeze-dried solid negative control and a virus RNA extraction reagent, wherein the freeze-dried solid RT-PCRmix contains a primer group corresponding to primer sequences of SARS-CoV-2 specific N gene, influenza A M gene, influenza B M gene and human endogenous reference gene RNAse P.

In some embodiments, the primer set is four sets of primers and probes, the sequences of which are as follows:

in some embodiments, the primers in the first combination are a 1 st primer set, the 1 st primer set for detecting a novel coronavirus 2019-nCoV; the primers in the second combination are a 2 nd primer group, and the 2 nd primer group is used for detecting the influenza A virus; the primers in the third combination are 3 rd primer groups, and the 3 rd primer groups are used for detecting the influenza B virus; the primers in the fourth combination are a 4 th primer group, and the 4 th primer group is used for detecting an internal standard gene RNaseP.

In some embodiments, the lyophilized solid RT-PCR Mix contains dATP, dGTP, dCTP, dUTP, Taq DNA polymerase, MMLV reverse transcriptase, RNase inhibitor, UDG enzyme, N gene-F/R/P, InfAM gene-F1/F2, InfAM gene-R1, InfAM gene R2, InfAM gene P, InfB M gene-F/R/P, RNase P-F/R/P, and a lyophilized complex protectant comprising 3% to 6% trehalose (w/v), 0.01% to 0.1% glycine (w/v), 0.1% to 1% bovine serum albumin (w/v), 1mmol/L-5mmol/LDTT, 0.1% to 0.3% Proclin 300, 0.1% to 1% formamide in a whole premix prior to lyophilization.

In some embodiments, the lyophilized solid RT-PCRMix is in a mixture in a full premix prior to lyophilization: the concentrations of the primer probes in the 1 st detection group are 200nM, 200nM and 100nM respectively, and the concentrations of the primer probes in the 2 nd detection group are 300nM, 300nM and 200nM respectively; the concentrations of the primer probes in the 3 rd detection group are respectively 500nM, 500nM and 300 nM; the concentration of the primer probe in the 4 th detection group is 500nM, 500nM and 600nM respectively.

In some embodiments, the liquid reconstitution Buffer comprises MgSO4, KCl, Tris-HCl, (NH4)2SO4, Triton X-100, and betaine.

In some embodiments, the lyophilized solid-state positive control is a lyophilized solid-state RNA synthesized in vitro and containing a SARS-CoV-2 specific gene N, an influenza a M gene, and a partial specific fragment of an influenza b M gene, and the lyophilized solid-state negative control is a lyophilized solid-state RNA corresponding to purified non-SARS-CoV-2N gene, an influenza a M gene, and an influenza b M gene.

The second aspect of the invention provides a triple fluorescence PCR rapid detection method for novel coronavirus, influenza A virus and influenza B virus, and the use method comprises the following steps:

(1) template RNA extraction, namely extracting RNA of a sample to be detected by adopting a nucleic acid extraction box;

(2) preparation of reagents: adding 15 μ L of reconstitution Buffer RB to each PCR tube containing the fully pre-mixed lyophilized particles;

(3) sample and control addition: adding 5 mul of extracted sample RNA, negative control and positive control into each tube according to the number of experimental samples;

(4) centrifuging and putting the mixture into a fluorescent quantitative PCR instrument for PCR amplification;

(5) and (6) judging the result.

In some embodiments, in the step (4), the PCR tube is placed on a suitable model fluorescent quantitative PCR instrument, and the fluorescent channels FAM, HEX, ROX, CY5 are selected; setting reaction conditions: reverse transcription at 50 deg.C for 30min, pre-denaturation at 95 deg.C for 3min, 3-step PCR amplification, fluorescence collection at 95 deg.C for 10sec, 55 deg.C for 10sec, and 72 deg.C for 60sec for 40 cycles.

In some embodiments, the specific step of determining the result in step (5) is: when the negative control has no expansion curve on FAM, HEX and ROX channels and the CT value of the positive control on FAM, HEX and ROX channels is less than 38, the test is established; when the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative; when the positive control and the negative control are established, the FAM channel positive is the novel coronavirus (SARS-CoV-2), the HEX channel positive is the influenza A virus, and the ROX channel positive is the influenza B virus.

Compared with the prior art, the invention has the beneficial effects that:

the detection method is convenient and rapid, has high sensitivity, strong specificity and low price, is suitable for popularization and application, and provides clinical auxiliary diagnosis for influenza A virus, influenza B virus and novel coronavirus (SARS-CoV-2); the kit combines a multiple fluorescent quantitative PCR technology and a freeze drying technology, uses 3 pairs of special primers and a human reference gene to amplify specific sequences of 3 pathogens in vitro, and combines a fluorescent probe to carry out real-time detection. The method is simple and convenient to operate, has low requirement on the operation level of detection personnel, can detect 3 common respiratory pathogens at one time, greatly saves the detection time and the detection cost, and realizes the rapid screening of mass samples. The kit can detect novel coronavirus, influenza A virus and influenza B virus simultaneously, the whole detection only needs 40 minutes to 1 hour, the result is accurate and reliable, and the specific expression is as follows:

1. the fluorescent quantitative PCR detection reagent combines the freeze-drying technology, and after the composite freeze-drying protective agent is added and the freeze-drying process is optimized, the stability of the reagent is greatly improved, the normal-temperature transportation is convenient, and the transportation cost is saved;

2. the RT-PCRMix freeze-drying reagent optimizes and matches specific primers and probes of 3 pathogens of human novel coronavirus, influenza A virus and influenza B virus, and ensures the detection efficiency of the 3 pathogens.

3. In the RT-PCRmix, the components in a primer, a probe, a reverse transcriptase, a PCR reaction enzyme and a PCR buffer system are uniformly mixed according to an optimized proportion and are subpackaged into a PCR tube, the RT-PCRmix can be used immediately after dissolving without independently adding the enzyme, repeated freezing and thawing and repeated subpackaging are avoided, the RT-PCRmix is simple, convenient and rapid to use and is more suitable for basic-level field detection;

4. meanwhile, the human-derived reference gene is detected, so that the quality of a clinical sample can be monitored, and the reliability of a detection result is improved.

5. The reaction needs a small amount of template, the sensitivity is high, and the lowest check limit can be as low as 10 copy numbers/Test.

6. Under the concentration range of 10copies, the detection sensitivity of the kit to the novel coronavirus, the influenza A virus and the influenza B virus is 100 percent, 95 percent and 99 percent respectively, and the detection specificity is 100 percent.

Drawings

FIG. 1 is a graph of the amplification curve of the sensitivity test of the complete premixed freeze-dried multiple fluorescent PCR analysis of the novel coronavirus, influenza A virus and influenza B virus.

FIG. 2 is a graph showing the specific test amplification curve of the complete premixed lyophilized multiplex fluorescence PCR analysis of the novel coronavirus, influenza A virus and influenza B virus.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.

The invention designs 8 specific primers and 4 probes according to the genome specificity of M gene of influenza A virus, M gene of influenza B virus, ORF1ab gene of novel coronavirus (2019-nCoV) and internal standard gene RNaseP. The primer group comprises four groups of primers and probes, and matched primers and probes for simultaneously detecting novel coronavirus, influenza A virus, influenza B virus and human-derived reference gene RNAse P. The sequences are shown in Table 1:

the primers in the first combination are a 1 st primer group, and the 1 st primer group is used for detecting the novel coronavirus 2019-nCoV; the primers in the second combination are a 2 nd primer group, and the 2 nd primer group is used for detecting the influenza A virus; the primers in the third combination are 3 rd primer groups, and the 3 rd primer groups are used for detecting the influenza B virus; the primers in the fourth combination are a 4 th primer group, and the 4 th primer group is used for detecting an internal standard gene RNaseP

The invention provides a triple fluorescence PCR rapid detection kit for novel coronavirus, influenza A virus and influenza B virus, which comprises a freeze-dried solid RT-PCR Mix, a liquid redissolving Buffer, a freeze-dried solid positive control, a freeze-dried solid negative control and a virus RNA extraction reagent

In the second aspect of the invention, a plurality of fluorescent quantitative PCR methods are established, real-time fluorescent PCR amplification technology is utilized, after optimization, fluorescent markers of the probes in the 1 st primer group to the 4 th primer group are respectively FAM, HEX, Rox and Cy5, and influenza A virus, influenza B virus and novel coronavirus (2019-nCoV) are distinguished according to amplification curves of different channels.

Example 1

The freeze-dried solid RT-PCR Mix contains primer groups and probes corresponding to primer sequences of an N gene of SARS-CoV-2, an influenza A M gene, an influenza B M gene and a human source internal reference gene RNAse P. Before freeze-drying, the solid RT-PCR Mix mainly contains specific primers and probes of N gene containing novel coronavirus (SARS-CoV-2), M gene containing influenza A virus, M gene containing influenza B virus and genome of internal standard gene RNaseP, dNTP, Taq DNA polymerase, reverse transcriptase, RNase inhibitor, UDG enzyme and freeze-drying composite protective agent, and the balance DEPC treatment water, and the specific components and content are shown in Table 2.

Table 2: novel coronavirus, influenza A virus and influenza B virus RT-PCRMix liquid

The preparation method of the freeze-dried solid-state novel coronavirus and influenza A and influenza B RT-PCR mix particles comprises the following steps:

1) primer synthesis: synthesizing oligonucleotide primers according to the matched primers and probe sequences of the novel coronavirus, influenza A virus, influenza B virus and human reference gene RNAse P, and quantitatively preparing;

2) preparing novel coronavirus, influenza A virus RT-PCR Mix liquid and influenza B virus RT-PCR Mix liquid: the relevant components requiring lyophilization were mixed under sterile conditions as shown in table 2.

3) Subpackaging: and subpackaging the product into 8 rows of PCR tubes by using a trace quick dispenser.

4) Half-covered PCR cover: the cover of the PCR tube is half-buckled on the PCR tube without fastening and compacting.

5) Vacuum freeze drying: pre-freezing for 3 hours at-35 ℃ in a freeze dryer. The pressure in the freeze dryer is reduced to below 10Pa and the vacuum treatment is carried out for 2 hours at the temperature of-35 ℃. The temperature inside the freeze dryer was raised to 10 ℃ under vacuum and vacuum-treated for 2 hours. The temperature inside the freeze dryer was raised to 30 ℃ under vacuum for 2 hours.

6) And (4) capping: and clicking an automatic button to execute capping operation after the freezing process is finished.

7) And (3) vacuum packaging: and packaging the capped product on a vacuum packaging machine by using an aluminum foil bag.

Secondly, preparing a liquid redissolving Buffer:

1) the liquid redissolving buffer comprises 3-10 mmol/L MgSO4, 7-13 mmol/L KCl, 13-22 mmol/L Tris-HCl, 8-15 mmol/L (NH4)2SO4, 0.1-0.3% Triton X-100 and 0.3-1.6 mol/L betaine.

2) Mixing the above components under sterile environment, and packaging into penicillin bottles;

3) and (4) capping: and (4) buckling the bottle cover on the penicillin bottle, and automatically pressing a button to perform the capping operation.

The positive control is lyophilized solid RNA corresponding to SARS-CoV-2N gene, influenza A M gene and influenza B virus M gene synthesized in vitro.

Preparation of lyophilized solid-state positive control RNA

1) In vitro synthesis of positive RNA fragment, firstly constructing positive control plasmid containing SARS-CoV-2N gene, influenza A M gene and influenza B virus M gene, and then constructing synthesized positive control plasmid 1: 10⁸Diluted to template, and primer pair 1, T7F 1 and T7R 1, T7F 1: TCACTATAGGGATGTCTGATAATGGA, T7R 1: AACATTGGCCGTGACAGCT, the first PCR amplification was performed. After identification as a specific product, the 1 st PCR product 1: 10⁵Double dilution, with primer pair 2T 7F 2 and T7R 1, T7F 2: TTCCGCATAATACGACTCACTATA, respectively; T7R 1: AACATTGGCCGTGACAGCTTGACA, performing a second PCR amplification, and purifying the PCR product of the 2 nd amplification with a column nucleic acid extraction kit. Then, using T7 RNA polymerase in vitro reverse transcription kit, the PCR product 1 after 2 nd amplification purification: 10³Diluting and carrying out reverse transcription. And after the reaction is finished, digesting by using DNase to remove redundant DNA, purifying the reverse transcription product by using the column type nucleic acid extraction kit, and obtaining the purified RNA which is the novel coronavirus RNA positive control.

2) Calibration of positive control RNA concentration: the concentration of the kit is measured to be 20-30 ng/mu L, according to the characteristics of the kit, the CT value is between 28 and 30, and PCR-grade water is used for carrying out the steps of 1: 10⁸And (5) diluting by times.

3) Subpackaging: and (3) subpackaging the diluted positive control nucleic acid into penicillin bottles.

4) Half plugging: the pipe cover is put on the pipe orifice without fastening and compacting.

Preparation of the lyophilized negative control:

the negative control is purified non SARS-CoV-2 specific gene N gene, influenza A M gene, influenza B virus M gene solid RNA. In the same way, a section of non-SARS-CoV-2 specific gene N, influenza A M gene and influenza B virus M gene solid RNA is synthesized in vitro by the method, diluted and freeze-dried, and stored at-20 ℃ for standby use, and the program is the same as the positive control.

Fourthly, assembling of the kit

1) Putting a vacuum aluminum foil bag of the novel coronavirus, influenza A virus and influenza B virus RT-PCRimixSARS-CoV-2 RT-PCR Mix into a waterproof and pressure-proof packaging box, and putting a penicillin bottle containing the positive control RNA freeze-dried particles and a penicillin bottle containing the negative control RNA freeze-dried particles into the packaging box.

2) Placing into a penicillin bottle for redissolving Buffer RB.

3) Put into the instruction book

4) And (6) packaging.

In some examples, viral RNA extraction reagents are also included. Comprises lysis solution, inhibitor removing solution, rinsing solution, eluent, etc. The lysis solution comprises 3-6M guanidine hydrochloride, 20-100 mM Tris-HCl, 5-30% Triton X-100 and the like, and the pH value is 6.0-6.8. The inhibitor removing solution contains 3-6M guanidine hydrochloride, 20-100 mM Tris-HCl, pH 6.0-6.8. The rinsing liquid contains 10-20 mM NaCl and 1-2 mM Tris-HCl, and has a pH value of 7-7.5. The eluent was PCR grade water.

EXAMPLE 2 method of handling detection kit

The operation method of the freeze-drying rapid fluorescence quantitative PCR detection kit for the novel human coronavirus, the influenza A virus and the influenza B virus comprises the following steps:

the method comprises the following steps:

(1) template RNA extraction, wherein a nucleic acid extraction box is adopted to extract RNA of a sample to be detected.

(2) Preparing a reagent: adding 15 mu L of redissolving Buffer RB into each PCR tube with the freeze-dried particles, and adding 50ul of redissolving Buffer RB into freeze-dried penicillin bottles of positive control and negative control;

(3) sample and control addition: according to the number of experimental samples, 5ul of the extracted sample RNA is added into each PCR tube, 5ul of the redissolved negative and positive controls are added into the corresponding PCR tube, and the cover of the PCR tube is buckled.

(4) Centrifuging and putting the mixture into a fluorescent quantitative PCR instrument for PCR amplification: and (3) centrifuging the PCR tube for a short time, and then putting the tube into a fluorescent quantitative PCR instrument.

Setting PCR parameters: the FAM, HEX, ROX and Cy5 channels are simultaneously selected as instrument channels; the PCR amplification program is 15min at 50 ℃ and 3min at 95 ℃, and the cycle is once; the fluorescence was collected at 60 ℃ after 45 cycles of 95 ℃ for 15s and 60 ℃ for 60 s.

(5) And (5) judging a result: the test is established when the negative control has no expansion curve on FAM, HEX and ROX channels, and the positive control has CT value <38 on FAM, HEX and ROX channels. When the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative. When the positive control and the negative control are satisfied, the FAM channel positive is the novel coronavirus (2019-nCoV), the HEX channel positive is the influenza A virus, and the ROX channel positive is the influenza B virus. The specific detection results were determined according to table 1.

Table 1: novel judgment standard of coronavirus, influenza A virus and influenza B virus full-premix freeze-drying multiplex fluorescence PCR kit

Example 3 validation test

In some examples, the reaction solution of the reaction is: the concentrations of the primer probes of the first detection group are respectively 200nM, 200nM and 100nM, and the concentrations of the primer probes of the second detection group are respectively 300nM, 300nM and 200 nM; the concentrations of the primer probes in the third detection group are respectively 500nM, 500nM and 300 nM; the concentrations of the primer probes in the fourth detection group are respectively 500nM, 500nM and 600 nM; the concentrations of the primer probes in the fifth detection group were 600nM, and 600nM, respectively.

The properties of the reagents of the invention are further illustrated below in connection with the experiments.

S1: linearity of assay sensitivity and amplification efficiency

Respectively extracting positive control nucleic acids of influenza A virus, influenza B virus and novel coronavirus (2019-nCoV) with nucleic acid extraction kit, wherein the concentrations are respectively 10⁷-10³copies/ml were assayed, 3 replicates per concentration. According to the analysis of the CT value of the PCR result, the slope of the plotted standard curve is-3.05, the logarithmic correlation R2 of the CT value to the initial template copy number is 0.9988, the amplification efficiency E is 110%, and the fluorescence PCR amplification graph is shown in fig. 1.

S2 minimum check Limit test for analytical sensitivity

Respectively extracting positive control nucleic acids of influenza A virus, influenza B virus and novel coronavirus (2019-nCoV) with nucleic acid extraction kit, wherein the concentrations are respectively 10⁷-10³copies/ml were assayed, 20 replicates per concentration. And calculating the positive detection rate, and calculating the result, wherein the minimum detection limit of the freeze-dried multiplex fluorescent PCR kit on the novel coronavirus, the influenza A virus and the influenza B virus is 10copies/ul, the analysis sensitivities are respectively 100%, 95% and 95%, and the detailed results are shown in tables 4-6.

Table 4: confirmation of minimum detection Limit for SARS-CoV-2

Table 5: identification of lowest detection limit for influenza A virus

Table 6: identification of lowest detection limit for influenza B virus

S3 assay specificity test

Respectively extracting positive controls of parainfluenza virus, adenovirus, mumps virus, respiratory syncytial virus, rhinovirus, coronavirus HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV by using a nucleic acid extraction kit, detecting by using a full-premix freeze-drying multiplex fluorescence PCR detection kit, and a fluorescence PCR amplification curve chart is shown in figure 2.

S4: clinical sample testing

1) Sample treatment, namely taking a sample to be detected, and extracting by using a virus RNA extraction kit to obtain a corresponding RNA detection sample;

2) adding 15 mu L of redissolving buffer into each PCR tube, and adding 5 mu L of the RNA detection sample when the dry powder in the tube is completely dissolved; another 2 tubes are taken as negative control and positive control, and corresponding control reagents of 5 mu L are added respectively.

3) Setting PCR reaction time and detection channels according to the instruction, and judging the result according to the judgment standard in the instruction after the reaction is finished.

The above description is only an example of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can make many variations and modifications of the present invention without departing from the scope of the present invention by using the method disclosed above, and the present invention is covered by the claims.

Claims

1. The triple fluorescence PCR rapid detection kit for the novel coronavirus, influenza A virus and influenza B virus is characterized in that: the kit comprises a freeze-dried solid RT-PCR Mix, a liquid redissolving Buffer, a freeze-dried solid positive control, a freeze-dried solid negative control and a virus RNA extraction reagent, wherein the freeze-dried solid RT-PCR Mix contains a primer group corresponding to primer sequences of SARS-CoV-2 specific N gene, influenza A M gene, influenza B M gene and human source internal reference gene RNAse P.

2. The triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 1, which is characterized in that: the primer group comprises four groups of primers and probes, and the sequences of the primers and the probes are as follows:

3. the triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 2, which is characterized in that: the primers in the first combination are a 1 st primer group, and the 1 st primer group is used for detecting SARS-CoV-2; the primers in the second combination are a 2 nd primer group, and the 2 nd primer group is used for detecting the influenza A virus; the primers in the third combination are 3 rd primer groups, and the 3 rd primer groups are used for detecting the influenza B virus; the primers in the fourth combination are a 4 th primer group, and the 4 th primer group is used for detecting an internal standard gene RNaseP.

4. The triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 1, which is characterized in that: the freeze-dried solid RT-PCR Mix contains dATP, dGTP, dCTP, dUTP, Taq DNA polymerase, MMLV reverse transcriptase, RNase inhibitor, UDG enzyme, N gene-F/R/P, InfA M gene-F1/F2, InfA M gene-R1, InfA M gene R2, InfA M gene P, InfB M gene-F/R/P, RNase P-F/R/P and a compound protective agent containing 3-6% trehalose (w/v), 0.01-0.1% glycine (w/v), 0.1-1% bovine serum albumin (w/v), 1-5 mmol/L DTT, 0.1-0.3% Proclin 300 and 0.1-1% formamide in a fully premixed mixed solution before freeze-drying.

5. The triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 4, wherein: the lyophilized solid RT-PCRMix is added in the mixed solution in the full premix before lyophilization: the concentrations of the primer probes in the 1 st detection group are 200nM, 200nM and 100nM respectively, and the concentrations of the primer probes in the 2 nd detection group are 300nM, 300nM and 200nM respectively; the concentrations of the primer probes in the 3 rd detection group are respectively 500nM, 500nM and 300 nM; the concentration of the primer probe in the 4 th detection group is 500nM, 500nM and 600nM respectively.

6. The triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 1, which is characterized in that: the liquid reconstitution Buffer contains MgSO4, KCl, Tris-HCl, (NH4)2SO4, Triton X-100 and betaine.

7. The triple fluorescent PCR rapid assay kit for the novel coronavirus, influenza A virus and influenza B virus according to claim 1, which is characterized in that: the freeze-dried solid positive control is a freeze-dried powdery solid RNA which is synthesized in vitro and contains specific segments of SARS-CoV-2 specific gene N, influenza A M gene and influenza B M gene; the freeze-dried solid negative control is freeze-dried solid RNA corresponding to purified non-SARS-CoV-2N gene, influenza A M gene and influenza B M gene.

8. The triple fluorescence PCR rapid assay method for novel coronavirus, influenza A virus and influenza B virus according to claim 1, characterized in that: the using method comprises the following steps:

(5) and (6) judging the result.

9. The triple fluorescence PCR rapid assay method for novel coronavirus, influenza A virus and influenza B virus according to claim 1, wherein: placing the PCR tube on a fluorescent quantitative PCR instrument with a proper model in the step (4), and selecting fluorescent channels FAM, HEX, ROX and CY 5; setting reaction conditions: reverse transcription at 50 deg.C for 30min, pre-denaturation at 95 deg.C for 3min, 3-step PCR amplification, fluorescence collection at 95 deg.C for 10sec, 55 deg.C for 10sec, and 72 deg.C for 60sec for 40 cycles.

10. The triple fluorescence PCR rapid assay method for novel coronavirus, influenza A virus and influenza B virus according to claim 1, which comprises: the specific steps of the result judgment in the step (5) are as follows: when the negative control has no expansion curve on FAM, HEX and ROX channels and the CT value of the positive control on FAM, HEX and ROX channels is less than 38, the test is established; when the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative; when the positive control and the negative control are satisfied, the FAM channel positive is the novel coronavirus (2019-nCoV), the HEX channel positive is the influenza A virus, and the ROX channel positive is the influenza B virus.