CN110760620A - Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method - Google Patents

Abstract

A swine fever virus and African swine fever virus double fluorescent PCR detection reagent, kit and detection method belong to the field of animal pathogen detection. The invention aims at the 5'-UTR gene of swine fever virus and the P72 gene of African swine fever virus, respectively designs and screens out primers and probes that can cover all strains and are suitable for double detection, including two pairs of specific primers and two specific primers. The present invention also describes a kit comprising the primers and probes and a PCR detection method using the primers and probes; the present invention, through the design of primers and the adjustment of each component of PCR, has different sensitivity and specificity. Under the premise of reducing the cost, the purpose of simultaneously detecting and distinguishing between swine fever virus and African swine fever virus can be realized in one analysis, which not only reduces the workload and cost of detection, but also greatly saves the time of detection and wins valuable time for epidemic prevention and control.

Description

A kind of swine fever virus and African swine fever virus double fluorescent PCR detection reagent and kit and detection methods

technical field

The invention belongs to the field of animal pathogen detection, and in particular relates to a swine fever virus and African swine fever virus double fluorescent PCR detection reagent, a kit and a detection method.

Background technique

Swine fever virus (Classical Swine Fever Virus, CSFV) belongs to Flaviviridae, Pestivirus genus. Swine fever is an acute, febrile, and highly contagious infectious disease characterized by systemic hemorrhage, necrosis, and infarction caused by high temperature and microvascular degeneration. Swine fever is extremely harmful to pigs and will cause heavy losses to the pig industry. African swine fever (ASF) is a severe infectious disease of warthogs, porcupines, wild boars and domestic pigs caused by African swine fever virus (ASFV). The virus belongs to the African swine fever virus family, the genus African swine fever virus, and is a linear double-stranded DNA virus. After pigs are infected with the virus, the main clinical symptoms are high fever, loss of appetite, skin cyanosis, and bleeding in lymph nodes, kidneys, and gastrointestinal mucosa after autopsy. up to 100%. The World Organization for Animal Health (OIE) lists swine fever and African swine fever as notifiable animal diseases. A class of animal diseases.

At present, the commonly used laboratory detection methods for African swine fever are mainly pathogenic detection. In terms of etiological diagnosis, virus isolation and identification is the gold standard for diagnosing animal diseases. However, because the isolation and culture of African swine fever requires a high level of laboratory, most places do not have this condition, so this method is difficult to popularize. Fluorescent PCR is a molecular biology technique used to amplify specific DNA fragments. It has the characteristics of high sensitivity, strong specificity, simplicity and rapidity, and low requirements for the purity of the specimen. It is currently the most widely used pathogenic detection technology. , On January 4, 2019, the China Center for Animal Disease Control and Prevention announced the evaluation results of the first batch of rapid detection reagents for African swine fever on-site. There are 11 varieties in 3 categories, of which 8 are fluorescent PCR products.

Although African swine fever has typical clinical symptoms and pathological changes, due to the increasingly complex clinical infection and mixed infection of various pathogens, it is difficult to diagnose African swine fever based on clinical symptoms and pathological changes. Large swine fever is even more indistinguishable. In addition, there is currently no vaccine that can effectively prevent African swine fever in the world. Therefore, for African swine fever, an accurate diagnosis can only be made as soon as possible, so that corresponding measures such as culling and disinfection can be taken in a timely manner. If a convenient and fast differential diagnosis method can be established, which can distinguish between swine fever virus and African swine fever virus, it will provide a scientific and accurate basis for the rapid diagnosis of swine fever and African swine fever in clinical practice, and then provide corresponding preventive measures. The formulation of control measures provides guidance, which will have huge market application prospects.

At present, fluorescent RT-PCR and fluorescent PCR methods and patents for detecting single swine fever virus and African swine fever virus have been reported, and dual fluorescent PCR detection reagents that can simultaneously detect swine fever virus and African swine fever virus, and their preparation method and application , also rarely reported.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide fluorescent PCR primers and probes for detecting swine fever virus and African swine fever virus, respectively.

The second object of the present invention is to provide a double fluorescent PCR detection kit for detecting and identifying swine fever virus and African swine fever virus.

The third object of the present invention is to provide a double fluorescent PCR detection method for identifying swine fever virus and African swine fever virus.

Compared with single-plex fluorescent PCR, the present invention realizes one analysis through primer design and adjustment of each component of PCR under the premise of not reducing sensitivity and specificity, and simultaneously detects and distinguishes swine fever virus and African swine fever virus. The purpose is not only to reduce the workload and cost of testing, but also to greatly save the time of testing and gain valuable time for epidemic prevention and control;

To achieve the above object, the technical scheme adopted by the present invention is as follows:

A swine fever virus and African swine fever virus dual fluorescent PCR detection reagent, for the 5'-UTR gene of swine fever virus and the African swine fever virus P72 gene, respectively design and screen out primers that can cover all strains and are suitable for dual detection and probes, including two pairs of specific primers and two specific probes, the lengths of the amplified target fragments are 94bp and 249bp respectively, and the primer and probe sequences are:

Swine fever virus:

Upstream primer CSFV-F: 5'-TGCCCAYAGTAGGACTAG-3'SEQ ID NO.1

Downstream primer CSFV-R: 5'-CTACTGACGACTGTCCTG-3'SEQ ID NO.2

Probe CSFV-P: 5'-TGGCGAGCTCCCCTGGGTGGTCTA-3'SEQ ID NO.3

African swine fever virus:

Upstream primer ASFV-F: 5'-GATACCACAAGATCTGCCGT-3'SEQ ID NO.4

Downstream primer ASFV-R: 5'-CTGCTCATGGTATCAATCTTATCG-3'SEQ ID NO.5

Probe ASFV-P: 5'-CCACGGGAGGAATACCAACCCAGTG-3'SEQ ID NO.6

The probe CSFV-P and the probe ASFV-P are respectively labeled with different fluorescent reporter groups at the 5' end, and the probe CSFV-P and the probe ASFV-P are labeled with the same or different fluorescence at the 3' end Quenching group, Y in the upstream primer CSFV-F is a degenerate base, Y=C/T.

Further, the fluorescent reporter group is FAM or VIC, and the fluorescence quenching group is TAMRA or BHQ.

Further, the probe CSFV-P is labeled with FAM at the 5' end and TAMRA at the 3' end; the probe ASFV-P is labeled with VIC at the 5' end and BHQ at the 3' end.

A double fluorescent PCR detection kit for swine fever virus and African swine fever virus includes the two pairs of specific primers and two specific probes.

Further, the kit also includes reaction buffer, enzyme, positive control and negative control.

Further, the reaction buffer is One Step RT-PCRbuffer; the enzymes are Ex Taq and PrimeScript RT Enzyme; the positive control is an in vitro recombinant plasmid of swine fever virus and African swine fever virus synthetic fragments; the negative control Deionized water for sterilization.

A double fluorescent PCR detection method for swine fever virus and African swine fever virus, comprising the following steps:

Step 1: Extract the DNA and RNA of the sample to be tested, and reverse-transcribe the RNA into cDNA;

Step 2, take the DNA and cDNA described in step 1 as templates, utilize the test kit of claim 4 to carry out double fluorescent PCR amplification;

Step 3: Analyze the PCR product, and determine whether the sample to be tested contains swine fever virus and/or African swine fever virus according to the results of the amplification reaction.

Further, in step 2, the final concentration of the optimal upstream primer CSFV-F and the downstream primer CSFV-R of the 5'-UTR gene of swine fever virus are both 0.4 μmol/L, and the final concentration of the optimal probe CSFV-P is 0.6 μmol/L; the final concentration of the optimal upstream primer ASFV-F and downstream primer ASFV-R for detection of African swine fever virus P72 gene was 0.6 μmol/L, and the final concentration of the optimal probe ASFV-P was 0.6 μmol/L.

Further, in step 2, the reaction procedure of the double fluorescent PCR amplification is: 42°C for 5 min, 95°C for 10s; 95°C for 5s, 60°C for 30s, a total of 45 cycles.

The beneficial effects of the present invention relative to the prior art are:

(1) Good coverage and versatility: The primers and probes involved in the present invention are designed for the most conserved genes of swine fever virus and African swine fever virus, respectively, covering all sequences of the existing virus in Genebank, not There will be missed inspections. In addition, the amplification conditions formed by the primers and probes of the present invention are the same as the amplification conditions of other important swine infectious diseases, and experiments can be carried out on the same platform with other diseases at the same time, and the versatility is good. The purpose of the initial research and development direction is to make diagnosis faster and more convenient; this is the main innovation of the present invention.

(2) Multiplex and high-throughput: The dual fluorescent PCR system can realize rapid identification and detection of swine fever virus and African swine fever virus in one reaction, and the detection can be completed within 65 minutes, saving detection time and cost . It can meet the requirements of large-scale testing, which is conducive to the timely diagnosis of epidemic diseases, especially mixed infections, and wins precious time for epidemic prevention and control.

(3) High sensitivity: The sensitivity of the dual fluorescence PCR detection method is equivalent to the corresponding single-plex fluorescence PCR detection method, which overcomes the weakness of the current domestic commercialized multiple fluorescence PCR kits that reduce the sensitivity.

(4) Strong specificity: When designing primers and probes, the possible interference between pathogens has been fully considered. It has been proved by experiments that this dual fluorescent PCR is compatible with other important infectious diseases common to pigs, such as porcine reproductive and respiratory syndrome virus, There was no cross-reaction among porcine pseudorabies virus, porcine foot-and-mouth disease virus, porcine circovirus type 2, porcine parvovirus, porcine Japanese encephalitis virus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus.

(5) The number of amplification cycles of the present invention is increased from 40 cycles commonly used in domestic kits to 45 cycles commonly used in imported kits, and the result determination standard is increased from 35 cycles commonly used to 38 cycles, so that the overall performance of the kit is increased. The level has been further improved; this is also one of the innovative points of the present invention.

Description of drawings

Fig. 1 is the gene-specific amplification curve of swine fever virus and African swine fever virus in the present invention. Among them, 1 is the amplification curve of swine fever virus positive control; 2 is the amplification curve of African swine fever virus positive control; 3-11 are negative control, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, swine foot-and-mouth disease virus, pig Amplification curves of circovirus type 2, porcine parvovirus, porcine Japanese encephalitis virus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus.

Fig. 2 is the gene amplification curve of swine fever virus in the present invention.

Figure 3 is the standard curve of the swine fever virus gene in the present invention.

Fig. 4 is the gene amplification curve of African swine fever virus in the present invention.

Figure 5 is the standard curve of African swine fever virus gene in the present invention.

Figure 6 is a comparison of the single-plex and double-fluorescence PCR amplification curves of the swine fever virus gene in the present invention. Among them, 1 is the amplification curve of swine fever virus single-plex fluorescence PCR; 2 is the amplification curve of swine fever virus in double fluorescence PCR of swine fever virus and African swine fever virus.

Figure 7 is a comparison of the single-plex and double-fluorescence PCR amplification curves of the African swine fever virus gene in the present invention. Among them, 1 is the amplification curve of ASFV single-plex fluorescence PCR; 2 is the amplification curve of ASFV in double fluorescence PCR of swine fever virus and ASFV.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall cover within the scope of the present invention. The methods used in the following examples are conventional methods unless otherwise specified, and the test materials used are conventional biochemical reagents unless otherwise specified.

Example 1:

Development of a dual fluorescent PCR assay for detection and identification of swine fever virus and African swine fever virus.

The first step is the design and synthesis of swine fever virus and African swine fever virus primers and probes:

On the basis of NCBI sequence alignment analysis, for the 5'-UTR gene of swine fever virus and the P72 gene of African swine fever virus, primers and probes that can cover all strains and are suitable for double detection were designed and screened respectively, including two For specific primers and two specific probes, the lengths of the amplified target fragments are 94bp and 249bp, respectively. The sequences of the primers and probes are as follows:

Swine fever virus:

Upstream primer: F: 5'-TGCCCAYAGTAGGACTAG-3'SEQ ID NO.1

Downstream primer: R: 5'-CTACTGACGACTGTCCTG-3'SEQ ID NO.2

Probe: P: 5'-TGGCGAGCTCCCCTGGGTGGTCTA-3'SEQ ID NO.3

The probe is labeled with FAM at the 5' end and TAMRA at the 3' end.

African swine fever virus:

Upstream primer: F: 5'-GATACCACAAGATCTGCCGT-3'SEQ ID NO.4

Downstream primer: R: 5'-CTGCTCATGGTATCAATCTTATCG-3'SEQ ID NO.5

Probe: P: 5'-CCACGGGAGGAATACCAACCCAGTG-3'SEQ ID NO.6

The probe is labeled with VIC at the 5' end and BHQ at the 3' end.

The second step is the preparation of swine fever virus and African swine fever virus positive controls:

(1) On the NCBI website, compare the 5'-UTR gene sequences of swine fever virus, select a fragment with a conservative sequence and include the above primers, and send the fragment to Sangon Bioengineering (Shanghai) Co., Ltd. for Synthesize; connect the synthesized fragment with the cloning vector, transform it into DH5α Escherichia coli genetically engineered bacteria, pick and extract the positive cloned plasmid; convert it into copy number according to the formula, and obtain the swine fever virus positive control with known copy number concentration.

(2) On the NCBI website, compare the gene sequences of African swine fever virus P72, select a fragment with a conservative sequence and include the above primers, and send the fragment to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis; Connect the synthesized fragment to the cloning vector, transform it into DH5α Escherichia coli genetically engineered bacteria, pick and extract the positive cloned plasmid; convert it into copy number according to the formula, and obtain the African swine fever virus positive control with known copy number concentration.

The third step is to optimize the double fluorescent PCR reaction system and amplification conditions for detecting the 5'-UTR gene of swine fever virus and the P72 gene of African swine fever virus:

(1) The concentrations of primers and labeled probes determined in the first step were diluted with sterile water to the final concentrations of 0.2 μmol/L, 0.4 μmol/L, 0.6 μmol/L, 0.8 μmol/L, 1.0 μmol/L, respectively. L and 1.2 μmol/L.

(2) Using the One Step PrimeScript RT-PCR Kit reagent and the recommended reaction amplification conditions, the fluorescent RT-PCR matrix screening test was carried out on the ABI7500 PCR instrument for primers and labeled probes with different concentration combinations.

(3) Obtain the minimum Ct value, the higher fluorescence intensity increase value (ΔRn) and the typical S-shaped amplification curve as the comprehensive judgment basis, and finally determine the optimal upstream primer and downstream primer for the detection of the 5'-UTR gene of swine fever virus The final concentration of the optimal probe is 0.4 μmol/L, and the final concentration of the optimal probe is 0.6 μmol/L; the final concentration of the optimal upstream and downstream primers for the detection of African swine fever virus P72 gene is 0.6 μmol/L, and the optimal probe concentration is 0.6 μmol/L. The final needle concentration was 0.6 μmol/L.

(4) Under the conditions of the optimal primer and probe concentration and the general fluorescent RT-PCR reaction amplification reagent, the addition amount of the reaction buffer in the amplification system was 6 μL, 8 μL, 10 μL and 12 μL in turn. Screening was performed using the amount to obtain the minimum Ct value, higher fluorescence intensity increase value (ΔRn) and typical S-shaped amplification curve as the comprehensive judgment basis, and the final addition amount of reaction buffer was determined to be 10 μL.

(5) Under the conditions of the optimal primer probe concentration and the optimal reaction buffer addition amount and the general fluorescent RT-PCR reaction amplification reagent, the addition amount of enzyme in the amplification system was 0.6 μL, 0.8 μL, 1.0 μL and 1.0 μL. 1.2μL, the amount of enzyme used was screened to obtain the minimum Ct value, higher fluorescence intensity increase value (ΔRn) and typical S-shaped amplification curve as the comprehensive judgment basis, and the final addition amount of enzyme was determined to be 1.0μL.

(6) Under the conditions of the optimal primer probe concentration, optimal reaction buffer, enzyme addition amount and general fluorescent RT-PCR reaction amplification reagent, the annealing extension temperature is in the range of 55℃--62℃. The annealing extension temperature was finally determined to be 60°C.

(7) In the optimized 20 μL PCR reaction system, the final concentrations of the upstream and downstream primers of swine fever virus were both 0.4 μmol/L, the final concentration of probes was 0.6 μmol/L, and the final concentrations of the upstream and downstream primers of African swine fever virus were both 0.4 μmol/L and 0.6 μmol/L. is 0.6 μmol/L, the final concentration of probe is 0.6 μmol/L, the amount of reaction buffer used is 10 μL, the amount of enzyme added is 1.0 μL, the amount of template added is 2.0 μL, and deionized water is added to the total volume of 20 μL . The optimized PCR amplification conditions were: 42°C for 5 min, 95°C for 10s; 95°C for 5s, 60°C for 30s, a total of 45 cycles.

(8) Result judgment

1) Quality control standard:

The positive control showed a specific sigmoid amplification curve, and the Ct value was around 20-25; the negative control had no amplification curve and no Ct value.

If this condition is satisfied, it is determined that the detection result is established.

2) Result judgment:

A. The sample to be tested has specific amplification curves in both the FAM and VIC channels, and the Ct value is ≤38, it is determined that both swine fever virus and African swine fever nucleic acid exist in the sample to be tested;

B. The sample to be tested has a specific amplification curve in a channel of FAM or VIC, and the Ct value is less than or equal to 38, it is determined that there is swine fever virus or African swine fever nucleic acid in the sample to be tested;

C. The sample to be tested has a specific amplification curve in the FAM and/or VIC channel, and 38 < Ct value ≤ 40, the test needs to be repeated, and the repeated result is positive, it is judged as positive, otherwise it is judged as negative;

D. There is no specific amplification curve in the FAM and VIC channels of the sample to be tested, and the Ct value is >40 or no Ct, it is determined that there is no swine fever virus or African swine fever nucleic acid in the sample to be tested.

The fourth step is the specificity experiment of swine fever virus and African swine fever virus double fluorescent PCR:

(1) Porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine foot-and-mouth disease virus, porcine circovirus type 2, porcine parvovirus, porcine Japanese encephalitis virus, porcine epidemic diarrhea virus, porcine infectious gastroenteritis Viruses are all commercially available vaccines. The extraction of the above-mentioned vaccine nucleic acid is carried out according to the instructions of the corresponding commercial kit;

(2) Fluorescent PCR amplification was carried out with the optimal reaction system and amplification conditions determined in the third step. The results showed that both swine fever virus and African swine fever virus positive control showed specific amplification curves; porcine reproductive and respiratory syndrome virus , porcine pseudorabies virus, porcine foot-and-mouth disease virus, porcine circovirus type 2, porcine parvovirus, porcine Japanese encephalitis virus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and negative controls did not appear to be specific. Amplification curve. The experimental results are shown in Figure 1.

The fifth step is the establishment of double fluorescent PCR sensitivity and standard curve for swine fever virus and African swine fever virus:

(1) Perform 10-fold serial dilution of the positive control of the 5'-UTR gene of CSFV, and perform fluorescence PCR amplification with the optimal reaction system and amplification conditions determined in the third step. The kinetic curve of fluorescence PCR amplification is shown in Fig. 2;

(2) Draw a standard curve according to the results of the amplification curve. The obtained regression equation of the standard curve is: Y=-3.17X+39.73, the correlation coefficient (R ² ) is 0.999, and the amplification efficiency is 106.8%, as shown in Figure 3; The designed primers and probes have high amplification efficiency and binding rate, and the optimized reaction conditions are suitable. 10 copies of the 5'-UTR gene positive control molecule of swine fever virus can be detected, which can be used for qualitative and quantitative detection of swine fever virus nucleic acid. ;

(3) 10-fold serial dilution of the African swine fever virus P72 gene positive control was performed, and fluorescence PCR amplification was performed with the optimal reaction system and amplification conditions determined in the third step. The kinetic curve of fluorescence PCR amplification is shown in Figure 4;

(4) Draw a standard curve according to the results of the amplification curve, and the obtained regression equation of the standard curve is: Y=-3.256+38.05, the correlation coefficient (R ² ) is 1, and the amplification efficiency is 102.8%, as shown in Figure 5; it shows that the designed The primers and probes have high amplification efficiency and binding rate, and the optimized reaction conditions are suitable. 10 copies of the positive control molecule of the African swine fever virus P72 gene can be detected, which can be used for qualitative and quantitative detection of African swine fever virus nucleic acid.

Example 2

Application of a dual fluorescent PCR assay for detection and identification of swine fever virus and African swine fever virus.

The detection and identification of swine fever virus and African swine fever virus double fluorescent PCR method established according to the present invention was compared with the previously established single-plex fluorescent PCR detection method of swine fever virus and African swine fever virus, and the recombinant plasmids of the two viruses with different concentrations were compared The combination is used as a clinical sample (sample number is: 1-12), and sterile water is used as a negative control, and the method provided by the present invention and the single-plex fluorescence PCR method are respectively used for laboratory detection. The results are shown in Table 1, Figure 6 and Figure 7 . The results show that the fluorescence intensity of swine fever virus single-plex fluorescence PCR is higher than that of swine fever virus and African swine fever virus double fluorescence PCR, the results are shown in Figure 6 (for a clearer comparison, Figure 6 only shows The amplification results of the combination plasmid 7-12 swine fever virus); the average Ct value of the 12 plasmid combination samples differed by 0.05, and there was almost no difference. There is almost no difference between the fluorescence intensity of ASFV single-plex fluorescence PCR and the fluorescence intensity of ASFV in ASFV and ASFV double fluorescence PCR, the results are shown in Figure 7 (for a clearer comparison, Figure 7 The amplification results of the combination plasmids 1-6 of African swine fever virus are shown); the average Ct values of the 12 plasmid combination samples differ by 0.32, which is also within the normal range of difference. These results show that compared with the corresponding single-plex fluorescent PCR detection method using the dual fluorescent PCR detection method for detecting and identifying swine fever virus and African swine fever virus provided by the present invention, there is no difference in sensitivity and characteristic, but the reaction time This is greatly shortened, thereby gaining valuable time for detection.

Table 1 The detection method of the present invention is compared with the Ct value result of the single-plex fluorescence PCR detection method

The above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the embodiments of the present invention. For those skilled in the art, other differences can also be made on the basis of the above-mentioned descriptions. Changes or changes in form cannot be exhaustively listed here, and all obvious changes or changes derived from the technical solutions of the present invention are still within the protection scope of the present invention.

sequence listing

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Heilongjiang Academy of Agricultural Sciences

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Claims (9)

1. A dual fluorescence PCR detection reagent for swine fever virus and African swine fever virus is characterized in that: aiming at the swine fever virus 5' -UTR gene and the African swine fever virus P72 gene, primers and probes which can cover all strains and are suitable for double detection are respectively designed and screened, the primers and probes comprise two pairs of specific primers and two specific probes, the lengths of amplified target fragments are respectively 94bp and 249bp, and the sequences of the primers and the probes are as follows:

hog cholera virus:

an upstream primer CSFV-F: 5 '-TGCCCAYAGTAGGACTAG-3'

The downstream primer CSFV-R: 5'-CTACTGACGACTGTCCTG-3'

Probe CSFV-P: 5'-TGGCGAGCTCCCTGGGTGGTCTA-3'

African swine fever virus:

an upstream primer ASFV-F: 5'-GATACCACAAGATCTGCCGT-3'

A downstream primer ASFV-R: 5'-CTGCTCATGGTATCAATCTTATCG-3'

Probe ASFV-P: 5'-CCACGGGAGGAATACCAACCCAGTG-3'

The probe CSFV-P and the probe ASFV-P are respectively marked with different fluorescent reporter groups at the 5 'end, and the probe CSFV-P and the probe ASFV-P are both marked with the same or different fluorescent quencher groups at the 3' end.

2. The reagent for detecting classical swine fever virus and African swine fever virus dual fluorescence PCR according to claim 1, wherein: the fluorescence reporter group is FAM or VIC, and the fluorescence quencher group is TAMRA or BHQ.

3. The reagent for detecting classical swine fever virus and African swine fever virus dual-fluorescence PCR according to claim 1 or 2, wherein: the probe CSFV-P is marked with FAM at the 5 'end and TAMRA at the 3' end; the probe ASFV-P is labeled with VIC at the 5 'end and BHQ at the 3' end.

4. A dual fluorescence PCR detection kit for classical swine fever virus and African classical swine fever virus is characterized in that: comprising two pairs of specific primers according to claim 1 or 2 or 3 and two specific probes.

5. The hog cholera virus-African hog cholera virus dual-fluorescence PCR detection kit according to claim 4, wherein: the kit also comprises reaction buffer, enzyme, positive control and negative control.

6. The hog cholera virus-African hog cholera virus dual-fluorescence PCR detection kit according to claim 5, wherein: the reaction buffer solution is One Step RT-PCRbuffer; the enzyme is Ex Taq and PrimeScriptRTenzyme; the positive control is in vitro recombinant plasmid of a swine fever virus and African swine fever virus synthetic fragment; the negative control was sterilized deionized water.

7. A double fluorescence PCR detection method for classical swine fever virus and African classical swine fever virus is characterized by comprising the following steps:

step one, extracting DNA and RNA of a sample to be detected, and carrying out reverse transcription on the RNA into cDNA;

step two, using the DNA and the cDNA in the step one as templates, and carrying out double fluorescence PCR amplification by using the kit of claim 4;

and step three, analyzing the PCR product, and judging whether the sample to be detected contains the classical swine fever virus and/or the African classical swine fever virus according to the amplification reaction result.

8. The swine fever virus and African swine fever virus dual-fluorescence PCR detection method according to claim 7, wherein: in the second step, the final concentrations of the most suitable upstream primer CSFV-F and the most suitable downstream primer CSFV-R of the hog cholera virus 5' -UTR gene are both 0.4 mu mol/L, and the final concentration of the most suitable probe CSFV-P is 0.6 mu mol/L; the final concentrations of the most suitable upstream primer ASFV-F and the downstream primer ASFV-R for detecting the African swine fever virus P72 gene are both 0.6 mu mol/L, and the final concentration of the most suitable probe ASFV-P is 0.6 mu mol/L.

9. The swine fever virus and African swine fever virus dual-fluorescence PCR detection method according to claim 7, wherein: in the second step, the reaction procedure of the double fluorescence PCR amplification is as follows: 5min at 42 ℃ and 10s at 95 ℃; 5s at 95 ℃ and 30s at 60 ℃ for 45 cycles.

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