Dual quantitative fluorescent primer probe composition, kit and identification method for identifying hog cholera virus

Abstract

The invention belongs to the technical field of animal pathogen molecular biology, and particularly relates to a dual quantitative fluorescent primer probe composition, a kit and an identification method for identifying hog cholera virus. Aiming at the current popular strains covering 3 genotypes, 11 gene subtypes and the like of the hog cholera gene, the invention selects 5UTR and NS3 gene sequences of hog cholera virus to design and synthesize specific primers and probes aiming at the two fragments; the specific primer has no non-specific amplification on African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type II, porcine epidemic diarrhea and bovine viral diarrhea. The invention establishes a method for identifying the classical swine fever virus wild strain and the vaccine strain and develops a kit convenient for diagnosis, and the kit and the identification method can simultaneously identify the classical swine fever virus wild strain and the vaccine strain, have the characteristics of high sensitivity, strong specificity, good repeatability and good stability, and have extremely strong application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal pathogen molecular biology, and particularly relates to a duplex fluorescent quantitative primer probe composition, kit and identification method for identifying wild strains and vaccine strains of porcine fever virus. BACKGROUND

[0002] Classical Swine Fever (CSF), also known as classical swine fever, is an acute and highly contagious disease caused by Classical Swine Fever Virus (CSFV). The virus belongs to the Flaviviridae family and the Pestivirus genus, has a single-stranded positive RNA genome of about 12.3 kb, encodes a polyprotein precursor, and is processed into four structural proteins (C, Erns, E1, E2) and eight non-structural proteins (such as NS2, NS3, NS5B, etc.) by host and viral proteases. Among them, the E2 protein is the main immunogenic protein that can induce neutralizing antibodies and is a key target for diagnosis and vaccine development.

[0003] The virus has a wide transmission route and can spread through direct contact, contamination (feed, water, equipment), and vertical transmission (placental infection). The virus is stable in the environment and sensitive to conventional disinfectants (such as sodium hydroxide and formalin). CSF is listed as a mandatory reporting disease by the World Organization for Animal Health (WOAH) and is classified as a category I animal disease in China, causing significant economic losses to the global pig industry. CSFV infects only a single host, i.e., pigs (domestic pigs and wild pigs), and is pathogenic to pigs. The incubation period is usually 5-15 days. The clinical manifestations include high fever (up to 42℃), hemorrhagic purpura on the skin, leukopenia, ataxia, abortion, and multiple organ hemorrhage. The disease course is short and the mortality rate is extremely high (up to 100% in acute cases). Due to the high similarity of symptoms to other pig diseases (such as African swine fever and swine erysipelas), clinical diagnosis requires laboratory detection.

[0004] At present, there are various methods for diagnosing swine fever, such as clinical and pathological observation, pathogenic diagnosis, serological diagnosis. Among them, the pathogenic diagnosis includes polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), isothermal amplification technology (RPA, LAMP). Serological diagnosis includes FAT, ELISA, IFA, IPT, DIA, IBT. Clinical and pathological observation does not need cost, but it is difficult to identify infectious diseases with similar symptoms. The isothermal amplification technology (RPA, LAMP) has high requirements for experimental conditions, and its sensitivity and effectiveness still need a large amount of clinical data verification. ELISA has the advantages of simple operation, low cost and short time, but the detection rate of chronic infection is not high. The serological methods such as IBT and IFA lack commercial kits. It can be seen that PCR and qPCR are the most reliable methods for diagnosing CSFV infection, and qPCR with high sensitivity is regarded as the gold standard.

[0005] Therefore, establishing a new CSFV fluorescent quantitative PCR detection method for detecting the target gene of CSFV is of great importance for early detection and prevention and control of the disease, in-depth study of the pathogenic mechanism of CSFV, and future identification of vaccine strains and wild virus infection. SUMMARY

[0006] Therefore, the first purpose of the present application is to provide a double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of swine fever virus, which has the characteristics of high sensitivity, strong specificity, good repeatability and stability;

[0007] The second purpose of the present application is to provide a double fluorescent quantitative kit for identifying wild strains and vaccine strains of swine fever virus.

[0008] The third purpose of the present application is to provide a double fluorescent quantitative PCR identification method for identifying wild strains and vaccine strains of swine fever virus.

[0009] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0010] A double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of swine fever virus comprises primer probe compositions for detecting 5' UTR fragment genes of conserved regions of swine fever virus and NS3 fragment genes, respectively; wherein,

[0011] The primer probe composition for detecting 5' UTR fragment genes of conserved regions of swine fever virus comprises:

[0012] The upstream primer CSFV-F1 has a nucleotide sequence as shown in SEQ ID NO: 1;

[0013] The nucleotide sequence of the downstream primer CSFV-R1 is shown in SEQ ID NO: 2.

[0014] The nucleotide sequence of the fluorescent probe CSFV-P1 is shown in SEQ ID NO: 3.

[0015] The primer probe composition for detecting the NS3 fragment gene of the porcine fever virus comprises:

[0016] The nucleotide sequence of the upstream primer CSFV-F2 is shown in SEQ ID NO: 4.

[0017] The nucleotide sequence of the downstream primer CSFV-R2 is shown in SEQ ID NO: 5.

[0018] The nucleotide sequence of the fluorescent probe CSFV-P2 is shown in SEQ ID NO: 6.

[0019] The nucleotide sequences of the 5` UTR fragment and the NS3 fragment genes of the porcine fever virus are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0020] Preferably, the probe is a hydrolysis probe.

[0021] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group and the 3' end is labeled with a quencher group.

[0022] More preferably, the fluorescent reporter group is selected from any one or combination of FAM, HEX, VIC; and the quencher group is selected from one or more of TAMARA, MGB.

[0023] More preferably, the 5' ends of different specific probes are labeled with different fluorescent reporter groups.

[0024] The present application also claims the use of the primer probe composition for preparing a duplex fluorescent quantitative PCR kit for identifying wild strains and vaccine strains of porcine fever virus.

[0025] The present application also discloses a duplex fluorescent quantitative PCR kit for identifying wild strains and vaccine strains of porcine fever virus, which comprises the primer probe composition.

[0026] Specifically, the kit further comprises a positive control and / or a negative control.

[0027] The positive control comprises a recombinant plasmid containing the 5` UTR fragment gene of the porcine fever virus and a recombinant plasmid containing the NS3 fragment gene of the porcine fever virus. Preferably, the negative control is water.

[0028] Specifically, the kit further comprises a PCR amplification solution and a fluorescence reaction solution. 2+ , and an enhancer and a stabilizer.

[0029] The application further discloses a duplex fluorescent quantitative PCR system for identifying wild strains and vaccine strains of CSFV, comprising the primer probe composition.

[0030] Preferably, in the primer probe composition, the primer of the 5' UTR fragment gene has a final concentration of 0.001-0.01 pmol / ul in the system, and more preferably 0.005 pmol / ul; and the probe has a final concentration of 0.01-0.02 pmol / ul in the system, and more preferably 0.0125 pmol / ul.

[0031] Preferably, in the primer probe composition, the primer of the NS3 fragment gene has a final concentration of 0.001-0.01 pmol / ul in the system, and more preferably 0.005 pmol / ul; and the probe has a final concentration of 0.01-0.02 pmol / ul in the system, and more preferably 0.0125 pmol / ul.

[0032] The application further discloses a method for using the primer probe composition, the kit or the fluorescent quantitative PCR system for a purpose other than disease diagnosis and / or treatment, which comprises the steps of performing real-time fluorescent quantitative PCR reaction on a sample to be tested by using the primer probe composition, the kit or the fluorescent quantitative PCR system, and collecting fluorescent signals for identification, so as to determine whether the sample contains wild strains and / or vaccine strains of CSFV.

[0033] Preferably, the real-time fluorescent quantitative PCR reaction condition comprises 95 DEG C for 30 s, 95 DEG C for 10 s, 58 DEG C for 20 s, and 45 cycles.

[0034] The application further discloses a use of the primer probe composition, the kit or the fluorescent quantitative PCR system for preparing a diagnostic reagent for a CSFV-related disease.

[0035] The double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of porcine fever virus provided by the application is targeted to porcine fever wild strains, includes 3 genotypes and 11 gene subtypes, i.e., 1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3 and 3.4, the primer probe of the conserved region of the wild strain and vaccine strain of porcine fever virus and the primer probe of the conserved region of the different subtypes of porcine fever virus are respectively determined through sequence alignment, and the primer probe sequences with close amplification efficiency are screened out through optimization, and the two pairs of fluorescent probes are further kept at a consistent amplification efficiency by adjusting the proportion of the primer and the fluorescent probe.

[0036] The double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of porcine fever virus provided by the application is designed for the primers / probes of two genes of 5' UTR and NS3, wherein the 5' UTR gene is the first choice for detecting porcine fever virus, and the gene is a relatively conserved gene of porcine fever virus; the NS3 region exhibits the characteristics of partial relative conservation in the wild strains of porcine fever. Therefore, the double fluorescent quantitative PCR detection method is established to identify the wild strains and vaccine strains of porcine fever virus, can effectively diagnose whether the gene deletion strain is infected, effectively makes up the problem that the wild strain and vaccine strain of porcine fever virus cannot be distinguished when the wild strain and vaccine strain of porcine fever virus are co-infected, and has better accuracy and applicability.

[0037] In addition, the non-structural protein NS3 of porcine fever virus has the activities of serine protease, nucleoside triphosphatase and RNA helicase, is an essential protein for virus replication and proliferation. The NS3 protein is closely related to the CPE of the virus to the host, is a marker of viral pathogenicity, but the immunity to the CSFV NS3 protein cannot resist the infection of the pathogenic virus.

[0038] The double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of porcine fever virus provided by the application is designed for the 5' UTR and NS3 two gene fragments of CSFV, and the designed primers / probes have high conservation through sequence comparison of 15 CSFV gene sequences. Through optimization, the standard curve has a good linear relationship (R 2 >0.99), and the minimum detection concentration of the positive plasmid is 100 copies / μl. 1x10 6 -10 3The mixed plasmid with 10 copies / ul was used as the reaction template of double qPCR, the coefficient of variation (CV) of three repeats in the group and between groups was less than 2%, indicating that the method has good repeatability. Through the detection of samples of different genotype strains, the obtained results meet the expectation. Therefore, the established kit has great significance for the molecular diagnosis of swine fever pathogens and the identification of wild strains and vaccine strains.

[0039] The double fluorescent quantitative primer probe composition for identifying wild strains and vaccine strains of swine fever virus has strong specificity, and there is no non-specific amplification curve for African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type II virus, porcine epidemic diarrhea and bovine viral diarrhea, so that the accuracy of detection is ensured.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] The detection kit can quickly identify wild strains and vaccine strains of swine fever virus, and is suitable for the currently popular wild strains and vaccine strains of swine fever in China. The specific primers and probes for the two genes are designed and synthesized by selecting the common conservative region of the 5' UTR gene sequence of the wild strain and the vaccine strain of swine fever and the relatively conservative region of the NS3 gene sequence of the wild strain of swine fever, and a method for identifying the wild strain and the vaccine strain of swine fever virus is established. The kit and the identification method can identify the wild strain and the vaccine strain of swine fever virus at the same time, the primers of different genes do not interfere with each other, and have the characteristics of high sensitivity, strong specificity, good repeatability and stability. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 It is a fluorescence amplification curve diagram of the 5' UTR gene of swine fever virus (the curve from left to right represents the copy number of mixed positive plasmid, which is 10 8 -10 2 copies / μL);

[0043] Figure 2 It is a standard curve diagram of the 5' UTR gene of swine fever virus;

[0044] Figure 3 It is a fluorescence amplification curve diagram of the NS3 gene of swine fever virus (the curve from left to right represents the copy number of mixed positive plasmid, which is 10 8 -10 2 copies / μL);

[0045] Figure 4 It is a standard curve diagram of the NS3 gene of swine fever virus;

[0046] Figure 5Singleplex fluorescence amplification curve of porcine pestivirus 5' UTR gene (from left to right, the curve represents the copy number of positive plasmid in turn 10 8 -10 2 copies / μL);

[0047] Figure 6 Singleplex fluorescence amplification curve of porcine pestivirus NS3 gene (from left to right, the curve represents the copy number of positive plasmid in turn 10 8 -10 2 copies / μL);

[0048] Figure 7 Specificity experiment fluorescence amplification curve results; wherein, 1 is the amplification curve of porcine pestivirus vaccine strain, 2-6 are PRV, PEDV, PCV2, PRRSV, BVDV, negative control respectively;

[0049] Figure 8 Specificity experiment fluorescence amplification curve results (similar to Figure 7 , representing the amplification results of two groups of primers and probes respectively); wherein, 1 is the amplification curve of porcine pestivirus vaccine strain, 2-6 are PRV, PEDV, PCV2, PRRSV, BVDV, negative control respectively;

[0050] Figure 9 Fluorescence amplification curve of different subtypes of strains;

[0051] Figure 10 Fluorescence amplification curve of different subtypes of strains (similar to Figure 9 , representing the amplification results of two groups of primers and probes respectively);

[0052] Figure 11 Fluorescence amplification curve of clinical samples with known genotypes, wherein, 1 is a whole blood sample, 2-9 are spleen, lung, liver, anal swab, kidney, heart, oral swab, lymph node samples respectively, 10 is a tonsil sample, 11-12 are negative controls;

[0053] Figure 12 Fluorescence amplification curve of clinical samples with known genotypes (similar to Figure 11 , representing the amplification results of two groups of primers and probes respectively), wherein, 1 is a whole blood sample, 2-9 are spleen, lung, liver, anal swab, kidney, heart, oral swab, lymph node samples respectively, 10 is a tonsil sample, 11-12 are negative controls. DETAILED DESCRIPTION

[0054] In the following examples of the present application, the specific primers and probes for the two genes of the wild strain and vaccine strain of CSFV are designed and synthesized according to the 5' UTR and NS3 gene sequences of CSFV, which can identify the two genes of the wild strain and vaccine strain of CSFV simultaneously, and the primers of different genes do not interfere with each other, and have the characteristics of high sensitivity, strong specificity, good repeatability and stability.

[0055] In the following examples of the present application, the 5'-end of the fluorescent probe CSFV-P1 and CSFV-P2 can be labeled with any one of the fluorescent reporter groups FAM, VIC, Cy5 and HEX, and the 3'-end can be labeled with the fluorescent quenching group MGB or TAMRA.

[0056] The fluorescent probes CSFV-P1 and CSFV-P2 are labeled with FAM and HEX, respectively.

[0057] Specifically, the CSFV 5' UTR gene identification region sequence is as follows: TACAGGACAGTCGTCAGTAGTTCGArCCGCTAGGGTTAAGGTGTGTCTTACAGGACAGTCGTCAGTAGTTCGACGTGAGCAGAAGCCCACCTCGAGATGCTATGTGGACGAGGGCATGCCCAAGACACACCTTAACCCTAGCGG (SEQ ID NO: 7).

[0058] The primer and probe composition for detecting the CSFV NS3 gene comprises:

[0059] The upstream primer CSFV-F1 is 5'-TACAGGACAGTCGTCAGTAGTTCGA-3' (SEQ ID NO: 1);

[0060] The downstream primer CSFV-R1 is 5'-CCGCTAGGGTTAAGGTGTGTCT-3' (SEQ ID NO: 2);

[0061] The fluorescent probe CSFV-P1 is 6-FAM-CCCACCTCGAGATGCTATGTGGACGA-TAMRA (SEQ ID NO: 3).

[0062] Specifically, the piglet disease virus NS3 gene identification region sequence is as follows: CACCTGTGTGACAGCATCAGGrCCGACTACCCTTCCACTTGATGCACCTGTGTGACAGCATCAGGAACCCCGGCCTTCTTTGACCTCAAGAACCTCAAAGGCTGGTCAGGGCTACCGATATTTGAGGCATCAAGTGGAAGGGTAGTCGG (SEQ ID NO: 8);

[0063] The primer probe combination for detecting the piglet disease virus NS3 gene comprises:

[0064] The upstream primer CSFV-F2 is 5'-CACCTGTGTGACAGCATCAGG-3' (SEQ ID NO: 4);

[0065] The downstream primer CSFV-R2 is 5'-GCCTCGAATATCGGTAGCCCTGA-3' (SEQ ID NO: 5);

[0066] The fluorescence probe CCSFV-P2 is HEX-CAGCCCTTGAGGTTCTTG-MGB (SEQ ID NO: 6).

[0067] The application also provides a double fluorescence PCR method for identifying and detecting the piglet disease wild strain and vaccine strain using the special primer and probe combination in the following examples, and the method is specifically as follows:

[0068] (1) Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA for standby use;

[0069] (2) The reaction system is prepared as follows: sample DNA 2 μl, primer premix 0.4 μl, probe premix 0.5 μl, PCR amplification solution 10 μl, and ddH2O 7.1 μl;

[0070] (3) The amplification program is as follows: 37 ℃ 2 min, 95 ℃ 30 s, 95 ℃ 10 s, 61 ℃ 30 s (collecting fluorescence signal), a total of 45 cycles; wherein, there are two fluorescence channels, which are: reporter group "FAM" and quencher group "TAMRA"; reporter group "HEX" and quencher group "MGB";

[0071] (4) The result judgment is as shown in the following table 1: the Ct value of the positive control in two channels is less than 30 and there is a specific amplification curve, the negative control has no Ct value and no specific amplification curve, and both conditions are met to determine that the experimental result is correct.

[0072] Table 1 result judgment standard

[0073] In summary, the standard results in Table 1 above show that:

[0074] In the FAM channel, Ct value ≤ 37 and specific amplification curve are positive, 37 < Ct value < 40 and specific amplification curve are suspicious, and no Ct value or Ct value ≥ 40 are negative.

[0075] In the HEX channel, Ct value ≤ 38 and specific amplification curve are positive, 38 < Ct value < 40 and specific amplification curve are suspicious, and no Ct value or Ct value ≥ 40 are negative.

[0076] In the above results, if the porcine pestivirus nucleic acid is suspicious, the DNA needs to be re-sampled and extracted for re-inspection, Ct value ≤ 40 and specific amplification curve are positive, otherwise, they are negative.

[0077] When both genes are positive, it can be determined that the porcine pestivirus wild strain is determined. When 5' UTR is positive and NS3 is negative, it can be determined that the porcine pestivirus vaccine strain is determined. If 5' UTR and NS3 are both negative, it is determined that the porcine pestivirus negative sample is determined.

[0078] The present application also provides a kit prepared by using the two groups of primers and probes, which can be used to assist in identifying the porcine pestivirus wild strain and vaccine strain, and / or detecting whether the porcine pestivirus wild strain and / or vaccine strain is contained in the sample.

[0079] Specifically, the kit further comprises the following reagents: positive control, negative control, primer premix, probe premix, and PCR amplification liquid; wherein,

[0080] The positive control contains a recombinant plasmid containing CSFV 1 and CSFV 2 genes.

[0081] The negative control is ddH2O.

[0082] The primer premix includes CSFV-F1, CSFV-R1, CSFV-F2, and CSFV-R with an initial concentration of 10 μM.

[0083] The probe premix includes CSFV-P1 and CSFV-P2 with an initial concentration of 10 μM.

[0084] The PCR amplification liquid includes 2x Animal Detection U+ Probe qPCR Super Premix.

[0085] The following examples of the present application also provide a method for detecting whether a wild strain and a vaccine strain of classical swine fever virus are contained in a test sample, i.e., using the primer probe combination or the kit to perform PCR amplification reaction on the nucleic acid extracted from the test sample, collecting the fluorescence signal, and determining whether the wild strain and / or the vaccine strain of classical swine fever virus are contained in the test sample.

[0086] The technical solutions of the present application are described in detail below. The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0087] Example 1

[0088] Preparation of positive standard

[0089] CSFV1 and CSFV2 plasmids were synthesized and constructed by Beijing Junji Biological Technology Co., Ltd. The sequence of the target gene was referred to CSFV 2.1 strain (GenBank sequence number: JX218094), and the cloning vector was pUC57 with AMP resistance. The recombinant plasmids were named as pUC57-CSFV1 and pUC57-CSFV2, respectively.

[0090] After amplification by shaking in LB culture solution, 2 μl was taken as a PCR reaction template. A 20 μl system was used, and the reaction system included: PCR Mix 5 μl, upstream primer 0.4 μl, downstream primer 0.4 μl, bacterial solution 2 μl, and ddH2O 2.2 μl.

[0091] The PCR amplification program was as follows: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 15 s, and 72℃ for 15 s, for a total of 40 cycles; and then 72℃ for 10 min.

[0092] After amplification, all products were identified by agarose gel electrophoresis, and the band was consistent with the size of the target fragment.

[0093] Singleplex qPCR condition optimization

[0094] Roche LightCycler 480Ⅱfluorescence quantitative PCR instrument was used, 2xAnimal Detection U+Probe qPCR Super Premix (Vazyme) was selected as the premix required for the reaction, and the following primer / probe concentration and annealing temperature were optimized.

[0095] The 5' UTR gene related primers and probes are designed as follows: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

[0096] The NS3 gene related primers and probes are designed as follows: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6;

[0097] Determination of singleplex qPCR reaction condition and primers

[0098] The total system of each gene single fluorescent quantitative PCR is determined by optimization to be 20 μl, and the specific components are shown in Table 2 below.

[0099] Table 2 Single fluorescent quantitative PCR reaction system

[0100] According to the above system, the single fluorescent amplification curve of the porcine pestivirus 5' UTR gene and NS3 gene formed by selecting different primer and probe combinations is shown in Figs. Figure 5 , 6, respectively.

[0101] The single qPCR amplification of the above primer and probe combinations is carried out, and the 5' UTR gene amplification graph is Figure 5 , and the NS3 gene amplification graph is Figure 6 The amplification curves of the two pairs of genes are good, and can be determined as the primer and probe combinations for subsequent experiments.

[0102] Based on the amplification efficiency shown in Figs. Figure 5 and Figure 6 , the following optimized primer and probe combinations can be determined as the primers / probes for double fluorescent quantitative PCR:

[0103] 5' UTR: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 ( Figure 5 );

[0104] NS3: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 ( Figure 6 ).

[0105] Example 2

[0106] Preparation of positive standard

[0107] The preparation method of the positive standard sample described in this example is the same as that of Example 1.

[0108] Duplex qPCR condition optimization

[0109] Using a fluorescence quantitative PCR instrument, 2x Animal Detection U+ Probe qPCR Super Premix was selected as the premix required for the reaction, and the primer / probe concentration and annealing temperature were optimized according to the order of first optimizing single qPCR and then optimizing double qPCR.

[0110] Through the single optimization in Example 1 above, the combination of 5' UTR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3), NS3 (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6) was selected as the final primer / probe combination system.

[0111] The primer / probe concentration and annealing temperature were optimized at an annealing temperature of 59°C, 60°C, and 61°C using the matrix method, and the specific procedures are shown in Table 3 below.

[0112] Table 3 Double qPCR condition optimization

[0113] The final optimized total system was 20 μl, and the specific composition is shown in Table 4 below.

[0114] The amplification program was 37°C for 2 min, 95°C for 30 s, and the cycle was 95°C for 10 s, 61°C for 30 s (fluorescence signal acquisition), for a total of 45 cycles.

[0115] Table 4 Double qPCR reaction system

[0116] After measuring the concentrations of pUC57-CSFV-1 and pUC57-CSFV-2 using a microspectrophotometer and converting them to copy numbers, they were 5×10 11 copies / μl and 4×10 11 copies / μl, respectively.

[0117] Each recombinant plasmid was first diluted to 1×10 9 copies / μl, and then an equal volume was mixed to obtain a mixed plasmid in which each plasmid was 5×10 8 copies / μl. 8 The diluted mixed plasmid was diluted by 10-fold to 1×10 0 copies / μl for constructing the standard curve. The specific operation process used the reaction conditions and system described above.

[0118] In this embodiment, the porcine pestivirus 5' UTR gene, the fluorescence amplification curve of the NS3 gene and the standard curve are shown in Figures Figures 1-6 .

[0119] As shown in the results Figures 1-6 , in the dilution of the current concentration range, the template amount and the corresponding Ct value showed a good linear relationship, and the correlation coefficient R 2 was 0.9995, 0.995, respectively, and the minimum detection amount of the fluorescence quantitative PCR was 100 copies / μl. Therefore, the fluorescence quantitative PCR system established in the present application has high sensitivity.

[0120] Example 3 Reproducibility test

[0121] Using 1×10 6 -10 3 copies / μl of the positive plasmid as the template, the fluorescence quantitative PCR was carried out according to the reaction system and procedure for fluorescence quantification provided in Example 2, and three repetitions were set for each gradient to verify the reproducibility of the method.

[0122] As shown in the results in Table 5, the coefficient of variation (CV value) of the repeated experiments of the present application was all below 2%, indicating that the present application has good reproducibility.

[0123] Table 5 Statistical calculation table of the coefficient of variation (CV value) of the repeated experiments

[0124] Example 4 Specificity test

[0125] Using the preserved porcine pestivirus, porcine pseudorabies virus, porcine blue ear virus, porcine circular type II virus, porcine epidemic diarrhea and bovine viral diarrhea positive samples as the templates, the fluorescence quantitative PCR amplification was carried out using the primer and probe composition system optimized in Example 2 of the present application, and the results are shown in Figure 7 ,8.

[0126] As can be seen, the detection results of different signal channels in the system were all negative, indicating that the method has strong specificity and no cross-reaction with other main infectious agents.

[0127] Example 5 Detection of different gene subtype strains

[0128] Using known different gene subtype porcine pestivirus nucleic acids and plasmids, including 1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3 and 3.4 subtypes, as the templates, the fluorescence quantitative PCR amplification was carried out using the primer and probe composition system optimized in Example 2 of the present application, and the results are shown in Figure 9 ,10.

[0129] Clinical sample testing of Example 6

[0130] Clinical samples of known genotype included whole blood, liver, heart, spleen, lung, kidney, lymph node, tonsil, oral swab and anal swab of CSFV HuB (1.1) infected. Nucleic acid was extracted from each of the 10 samples and evaluated by duplex qPCR. The clinical samples were tested using the duplex qPCR system as established in Example 2 and the expected amplification was observed for each sample, as shown in Table 12. Figure 11

[0131] It can be seen that the duplex fluorescent quantitative PCR detection method established by the present application can be used to identify wild strains and vaccine strains of porcine fever virus, can effectively diagnose whether wild strains are infected, and has the advantages of strong detection and good specificity.

[0132] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.​

Claims

1. A duplex fluorescent quantitative primer probe composition for identifying wild strain and vaccine strain of swine fever virus, characterized in that, The primer probe composition comprises a primer probe composition for detecting a 5' UTR fragment gene of a conserved region of a classical swine fever virus and a primer probe composition for detecting a NS3 fragment gene of the classical swine fever virus, respectively. The primer probe composition for detecting the 5' UTR fragment gene of the conserved region of the classical swine fever virus comprises: The upstream primer CSFV-F1 has a nucleotide sequence as shown in SEQ ID NO: 1; The downstream primer CSFV-R1 has a nucleotide sequence as shown in SEQ ID NO: 2; The fluorescent probe CSFV-P1 has a nucleotide sequence as shown in SEQ ID NO: 3; The primer probe composition for detecting the NS3 fragment gene of the classical swine fever virus comprises: The upstream primer CSFV-F2 has a nucleotide sequence as shown in SEQ ID NO: 4; The downstream primer CSFV-R2 has a nucleotide sequence as shown in SEQ ID NO: 5; The fluorescent probe CSFV-P2 has a nucleotide sequence as shown in SEQ ID NO: 6; The nucleotide sequences of the 5' UTR fragment gene of the conserved region of the classical swine fever virus and the NS3 fragment gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

2. The primer probe composition of claim 1, wherein, The probe is a hydrolysis probe.

3. The primer probe composition of claim 2, wherein The 5' end of the probe is labeled with a fluorescent reporter group and the 3' end is labeled with a quencher group.

4. Use of the primer probe composition of any one of claims 1 to 3 in the preparation of a duplex fluorescent quantitative PCR kit for identifying wild strains and vaccine strains of a classical swine fever virus.

5. A duplex real-time PCR kit for differentiating between wild-type and vaccine strains of Pestivirus, characterized in that, The kit comprises the primer probe composition of any one of claims 1 to 3.

6. The kit of claim 5, wherein The kit further comprises a positive control and / or a negative control; the positive control comprises a recombinant plasmid containing the 5' UTR fragment gene of the conserved region of the classical swine fever virus and a recombinant plasmid containing the NS3 fragment gene of the classical swine fever virus; and the negative control is water.

7. A fluorescent quantitative PCR system for identifying wild-type and vaccine strains of swine fever virus, characterized by, The kit comprises the primer probe composition of any one of claims 1 to 3.

8. The fluorescent quantitative PCR system for identifying wild strain and vaccine strain of swine fever virus according to claim 7, characterized in that, In the primer probe composition for detecting the 5' UTR fragment gene of the conserved region of the classical swine fever virus, the final concentration of the primer for the 5' UTR fragment gene in the system is 0.001-0.01 pmol / μL, and the final concentration of the probe in the system is 0.01-0.02 pmol / μL; and in the primer probe composition for detecting the NS3 fragment gene of the classical swine fever virus, the final concentration of the primer for the NS3 fragment gene in the system is 0.001-0.01 pmol / μL, and the final concentration of the probe in the system is 0.01-0.02 pmol / μL.

9. A method for identification, which is not for the purpose of disease diagnosis and / or treatment, using the primer probe composition according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 6 or the fluorescent quantitative PCR system according to claims 7 to 8, characterized in that, The identification method comprises the steps of performing real-time fluorescent quantitative PCR reaction on a sample to be tested using the primer probe composition, the kit or the fluorescent quantitative PCR system, and detecting the fluorescence signal to determine whether the sample contains wild strains and / or vaccine strains of the classical swine fever virus.

10. Use of the primer probe composition of any one of claims 1 to 3 or the kit of any one of claims 5 to 6 or the fluorescent quantitative PCR system of claims 7 to 8 in the preparation of a diagnostic reagent for a classical swine fever virus related disease.

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