Duplex PCR (Polymerase Chain Reaction) primer and kit for identifying wild strain and vaccine strain of duck plague virus and application of duplex PCR primer and kit

Abstract

The invention belongs to the technical field of biology, and particularly relates to a duplex PCR primer and kit for identifying a duck plague virus wild strain and a vaccine strain and application of the duplex PCR primer, and the duplex PCR primer comprises a primer pair P1 and a primer pair P2 which are designed based on a conserved region in a 1002bp fragment of a duck plague virus UL2 gene; the primer pairs P3 and P4 are designed on the basis of a conserved region in a 1930bp fragment of the duck plague virus UL56-LORF5 gene. The duplex PCR primer group provided by the invention can be used for simultaneously distinguishing classic wild strains, variant wild strains and vaccine strains of the duck plague virus, is rapid and accurate, and has relatively high specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a double PCR primer and a kit for identifying a wild strain and a vaccine strain of duck plague virus and an application thereof. Background Art

[0002] Duck plague, also known as duck viral enteritis (DVE), is an acute contact infectious disease of ducks, geese, swans and other Anseriformes caused by duck plague virus (DPV). Its pathogenic characteristics are vascular damage, tissue bleeding, digestive tract and lymphatic organ damage. The disease can lead to a decrease in egg production and death of commercial waterfowl. It is distributed in duck-raising areas around the world. Its prevention and control are directly related to the sustainable and stable development of waterfowl farming. The prevention of duck plague in my country mainly adopts attenuated vaccines, and accurate diagnosis and monitoring are the basis and prerequisite for effective prevention and control of the disease. At present, there are many methods for the diagnosis and monitoring of DPV reported, such as immunofluorescence, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). However, in production practice, when these methods are used to detect wild strains of duck plague virus and duck plague vaccine strains, they all show positive results and are difficult to distinguish. Therefore, when diagnosing and monitoring duck plague virus in farms that have been immunized with duck plague virus, it is impossible to objectively describe the cause of the positive result. Summary of the invention

[0003] The purpose of the present invention is to provide a dual PCR primer, a kit and an application thereof for distinguishing wild strains and vaccine strains of duck plague virus, which can simultaneously distinguish the classical wild strains, variant wild strains and vaccine strains of duck plague virus.

[0004] The present invention provides a dual PCR primer set for distinguishing wild strains and vaccine strains of duck plague virus, the dual PCR primer set comprising:

[0005] Primer pair P1 and P2 designed based on the conserved region within the 1002 bp fragment of the duck plague virus UL2 gene;

[0006] Primer pair P3 and P4 were designed based on the internal conserved region of the 1930 bp fragment of the duck plague virus UL56-LORF5 gene.

[0007] As a preferred solution, the gene sequence of P1 is shown as SEQ ID NO.1;

[0008] The gene sequence of P2 is shown in SEQ ID NO.2;

[0009] The gene sequence of P3 is shown in SEQ ID NO.3;

[0010] The gene sequence of P4 is shown in SEQ ID NO.4.

[0011] The present invention provides the use of the above-mentioned dual PCR primer set in the preparation of a product for identifying wild strains and vaccine strains of duck plague virus.

[0012] The invention provides a kit for distinguishing wild strains and vaccine strains of duck plague virus, comprising the above-mentioned double PCR primer set and conventional PCR reagents.

[0013] The present invention provides a dual PCR detection method for identifying wild strains and vaccine strains of duck plague virus, comprising the following steps: extracting genomic DNA of a sample to be tested, performing dual PCR amplification using the dual PCR primer set or the dual PCR primer set in the kit, and identifying the type of the sample according to the amplification result.

[0014] As a preferred embodiment, the sample types include: one or more of the classical wild strains, variant wild strains and vaccine strains of duck plague virus.

[0015] As a preferred solution, when the PCR amplification band obtained by using primer pair P1 and P2 is 335 bp, and no band is amplified by primer pair P3 and P4, the sample to be tested contains duck plague vaccine strain;

[0016] When the PCR amplification band obtained by primer pair P1 and P2 is 862 bp, and the PCR amplification band obtained by primer pair P3 and P4 is 507 bp, the sample to be tested contains the duck plague classic wild strain;

[0017] When the PCR amplification band obtained by using primer pair P1 and P2 is 335 bp, and the PCR amplification band obtained by using primer pair P3 and P4 is 507 bp, the sample to be tested contains a mutant wild strain of duck plague.

[0018] As a preferred solution, the reaction system of the double PCR amplification is 25 μL, including: 2×PCRMix 12.5 μL, 1.0 μL each of P1, P2, P3 and P4, 2.0 μL of genomic DNA of the sample to be tested and the balance of ddH2O water.

[0019] As a preferred embodiment, the reaction procedure of the double PCR amplification includes: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 7 min, and storage at 4°C.

[0020] The present invention provides the use of the above-mentioned dual PCR primer set or the above-mentioned kit or the above-mentioned dual PCR detection method in distinguishing wild strains and vaccine strains of duck plague virus

[0021] Beneficial effect: The present invention provides a dual PCR primer set for distinguishing wild strains and vaccine strains of duck plague virus, the dual PCR primer set comprising: primer pairs P1 and P2 designed based on the conserved region inside the 1002bp fragment of duck plague virus UL2 gene, and primer pairs P3 and P4 designed based on the conserved region inside the 1930bp fragment of duck plague virus UL56-LORF5 gene. The dual PCR primer set provided by the present invention can simultaneously distinguish between the classical wild strains, variant wild strains and vaccine strains of duck plague virus, and is fast and accurate, with high specificity and sensitivity.

[0022] The present invention provides a dual PCR detection method for identifying wild strains and vaccine strains of duck plague virus, comprising the following steps: extracting genomic DNA of the sample to be tested, performing dual PCR amplification using the dual PCR primer set, and identifying the type of sample according to the amplification result; that is, the duck plague virus classic wild strain, variant wild strain and vaccine strain can be distinguished according to the PCR amplification result. The dual PCR detection method provided by the present invention has the advantages of being simple, rapid, specific, sensitive and economical, and the result judgment method is simple, suitable for use in scientific research, clinical and grassroots laboratories, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a comparison of the genome sequences of duck plague virus vaccine strain, classical wild strain and variant wild strain, and a schematic diagram of primer positions;

[0024] Figure 2 This is the electrophoresis diagram of double PCR detection of duck plague virus classic wild strain, variant wild strain and vaccine strain; where M: DL2000 DNA ladder; lane 1: PCR amplification product using duck plague virus vaccine strain DNA as template, lane 2: PCR amplification product using duck plague variant wild strain DNA as template, lane 3: PCR amplification product using duck plague classic wild strain DNA as template;

[0025] Figure 3 Electropherogram for specificity evaluation of dual PCR primer set;

[0026] Figure 4 This is the electrophoresis diagram of the sensitivity test of the double PCR primer set to detect the duck plague virus vaccine strain;

[0027] Figure 5 This is the electrophoresis diagram of the sensitivity test of the double PCR primer set to detect the classic wild strain of duck plague virus;

[0028] Figure 6 This is the electrophoresis diagram of the sensitivity test of the dual PCR primer set for detecting mutant wild strains of duck plague virus. DETAILED DESCRIPTION

[0029] The present invention provides a dual PCR primer set for distinguishing duck plague virus from vaccine strains, the dual PCR primer set comprising:

[0030] Primer pair P1 and P2 designed based on the conserved region within the 1002 bp fragment of the duck plague virus UL2 gene;

[0031] Primer pair P3 and P4 were designed based on the internal conserved region of the 1930 bp fragment of the duck plague virus UL56-LORF5 gene.

[0032] The present invention compares and analyzes the whole genome of wild strains of duck plague virus prevalent in the early stage with vaccine strains, and finds that there are differences between the classic wild strains and the vaccine strains in the UL2 gene (1002bp), and the vaccine strain UL2 gene has a 528bp deletion. This feature can be used as a method for distinguishing the classic wild strains from the vaccine strains. Using this target, duck plague viruses in recent years are detected, and it is found that the wild strain amplified fragment has a similar amplification result to the vaccine strain, and a 528bp deletion has also appeared in the UL2 gene, indicating that the virus has mutated, and is therefore called a mutant wild strain. Further analysis of the genome sequence of the mutant wild strains in recent years found that the genome of the popular mutant wild strains in recent years has mutated, and the same deletion as the vaccine virus has also appeared in the UL2 gene, resulting in the detection of the 528bp deletion in UL2 alone. The wild strain and the vaccine strain cannot be distinguished. In-depth analysis of the genome sequence found that there are differences between the classic wild strains and the mutant wild strains and the vaccine strains in the middle of the UL56 and LORF5 genes, and the vaccine strain has a 1930bp deletion, while the classic wild strains and the mutant wild strains do not have this part of the deletion. Among them, the genome sequence comparison and primer positions of duck plague virus vaccine strain, classic wild strain and variant wild strain are as follows Figure 1 shown.

[0033] In the present invention, the duck plague virus UL2 gene was selected, and specific primer pairs P1 and P2 were designed and synthesized at both ends of the 528 bp fragment missing from the vaccine strain. The sequences of the specific primer pairs are as follows:

[0034] Upstream primer P1 (SEQ ID NO. 1): 5′-CGGTTTGAACGCTTCCTGAG-3′;

[0035] Downstream primer P2 (SEQ ID NO. 2): 5′-AACGAGGGAGACCCAAATGAC-3′;

[0036] The classic and variant duck plague virus UL56-LORF5 genes were selected, and specific primer pairs P3 and P4 were designed and synthesized within the 1930 bp fragment. The sequences of the specific primer pairs are as follows:

[0037] Upstream primer P3 (SEQ ID NO. 3): 5′-CAACCCACAGTGATGAAAGAA-3′;

[0038] Downstream primer P4 (SEQ ID NO. 4): 5'-CAAATCAAACGGACTGACAAA-3'.

[0039] As a specific implementation method, the genomic DNA of the classic wild strain, variant wild strain and vaccine strain of duck plague virus can be extracted respectively; double PCR amplification is performed using specific primer pairs P1 and P2 and specific primer pairs P3 and P4, and negative and blank controls are set up at the same time;

[0040] The reaction system was as follows: 2×PCRMix 12.5 μL, specific primers P1, P2, P3 and P4 1.0 μL each, duck plague virus genomic DNA 2.0 μL, and ddH2O water was used to make up to 25 μL;

[0041] Reaction parameters: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 7 min, stored at 4℃ for future use;

[0042] The PCR products obtained in the above steps were analyzed by 1% agarose gel electrophoresis.

[0043] As a specific implementation method, the following judgment is made based on the amplification results: if the PCR amplification band of the P1 and P2 primer pair is 335bp, and the PCR amplification band of the P3 and P4 primer pair is no band, then the sample to be tested contains the duck plague vaccine strain; if the PCR amplification band of the P1 and P2 primer pair is 862bp, and the PCR amplification band of the P3 and P4 primer pair is 507bp, then the sample to be tested contains the classic wild strain of duck plague; if the PCR amplification band of the P1 and P2 primer pair is 335bp, and the PCR amplification band of the P3 and P4 primer pair is 507bp, then the sample to be tested contains the variant wild strain of duck plague.

[0044] The present invention provides the use of the above-mentioned dual PCR primer set or the above-mentioned kit or the above-mentioned dual PCR detection method in distinguishing wild strains and vaccine strains of duck plague virus. The dual PCR detection method provided by the present invention has the advantages of being simple, rapid, specific, sensitive and economical, and the result judgment method is simple, suitable for use in scientific research, clinical and grassroots laboratories, and has broad application prospects.

[0045] To further illustrate the present invention, the dual PCR primers, kit and application thereof for distinguishing wild strains and vaccine strains of duck plague virus provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1 PCR detection method for distinguishing wild strains and vaccine strains of duck plague virus

[0047] The duck plague virus UL2 gene [GenBank accession no. JQ673560 (classic wild strain) and EU082088 (vaccine strain)] was selected, and specific primer pairs were designed and synthesized at both ends of the 528 bp fragment deleted in the vaccine strain. The sequences of the specific primer pairs are as follows:

[0048] Upstream primer P1 (SEQ ID NO. 1): 5′-CGGTTTGAACGCTTCCTGAG-3′;

[0049] Downstream primer P2 (SEQ ID NO. 2): 5'-AACGAGGGAGACCCAAATGAC-3'.

[0050] The UL56-LORF5 gene of the classical wild strain of duck plague virus (GenBank accession no. MN518864) and the variant wild strain (GenBank accession no. MH401586) was selected, and a specific primer pair was designed and synthesized within the 1930 bp fragment. The sequence of the specific primer pair is as follows:

[0051] Upstream primer P3 (SEQ ID NO. 3): 5′-CAACCCACAGTGATGAAAGAA-3′;

[0052] Downstream primer P4 (SEQ ID NO. 4): 5'-CAAATCAAACGGACTGACAAA-3'.

[0053] The genomic DNA of the classical wild strain, variant wild strain and vaccine strain of duck plague virus were extracted respectively.

[0054] Use specific primers P1 and P2 and specific primers P3 and P4 for double PCR amplification, and set up negative and blank controls. The reaction system is as follows:

[0055] 2×PCRMix 12.5μL, specific primers P1, P2, P3 and P4 1.0μL each, duck plague virus genomic DNA 2.0μL, add ddH2O water to make up to 25μL.

[0056] Reaction parameters: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles, and final extension at 72°C for 7 min. Store at 4°C for future use.

[0057] The PCR products obtained in the above steps were analyzed by 1% agarose gel electrophoresis. The experimental results are shown in Figure 2 .

[0058] Depend on Figure 2 It can be seen that the PCR amplification band of duck plague vaccine strain is 335bp (lane 1); the PCR amplification band of duck plague variant wild strain is 335bp and 507bp (lane 2); the PCR amplification band of duck plague classic wild strain is 862bp and 507bp (lane 3). The above results show that this method can well distinguish duck plague virus wild strain and vaccine strain infection.

[0059] Example 2 Specificity Evaluation Test

[0060] The above primers were used to perform PCR on DNA extracted from 13 common pathogens such as Newcastle disease virus (NDV), duck viral hepatitis virus (DHV), duck Tembusu virus (DTMUV), avian reovirus (ARV), infectious laryngotracheitis virus (ILTV), Marek's virus (MDV), new duck parvovirus (GPV), Muscovy duck parvovirus (MDPV), avian influenza (AIV), Riemerella anatipestifer (RA), duck Salmonella (Sa1.A), Pasteurella mitis (PM) and duck Escherichia coli (E.coli) according to conventional methods. At the same time, a positive control of duck plague virus vaccine strain, a positive control of duck plague virus variant wild strain, a positive control of duck plague virus classic wild strain and a blank control were set up. PCR detection was performed according to the reaction system and parameters of Example 1, and the obtained PCR products were analyzed by 1% agarose gel electrophoresis. The detection results of the specific experiment are shown in FIG. Figure 3 .

[0061] Depend on Figure 3 It can be seen that except for the duck plague virus vaccine strain positive control (lane 1), duck plague virus variant wild strain positive control (lane 2), duck plague virus classic wild strain positive control (lane 3) which showed specific amplification, 13 common pathogens including NDV (lane 4), DHV (lane 5), DTMUV (lane 6), ARV (lane 7), ILTV (lane 8), MDV (lane 9), GPV (lane 10), MDPV (lane 11), AIV (lane 12), RA (lane 13), Sal.A (lane 14), PM (lane 15) and E.coli. (lane 16) did not show amplification bands, which were negative. The blank control (lane 17) was also negative. The above results show that the PCR amplification method is highly specific.

[0062] Example 3 Sensitivity test

[0063] The above 862bp, 507bp, and 335bp amplified fragments were recovered from gel and cloned into the pMD19-T vector to construct recombinant plasmids pMD-862, pMD-507, and pMD-335. The positive recombinant plasmids were purified and the OD was measured. 260 nm, calculate the copy number of each plasmid, and then dilute the three recombinant plasmids 10-fold to 5×10 7 , 5×10 6 , 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 , 5×10 -1 copies / μL.

[0064] The sensitivity of the method was tested by simulating the amplification results of the duck plague virus vaccine strain: PCR amplification was performed using the above-mentioned 10-fold diluted pMD-335 as a template, PCR detection was performed according to the reaction system and parameters of Example 1, and the obtained PCR products were analyzed by 1% agarose gel electrophoresis. The results of the sensitivity experiment are shown in Figure 4 .

[0065] The sensitivity of the method was tested by simulating the amplification results of the classic wild strain of duck plague virus: pMD-862 and pMD-507 diluted 10 times as above were mixed in equal proportions according to the same dilution as templates for PCR amplification, and PCR detection was performed according to the reaction system and parameters of Example 1, and the obtained PCR products were analyzed by 1% agarose gel electrophoresis. The results of the sensitivity experiment are shown in Figure 5 .

[0066] The sensitivity of the method was tested by amplification of simulated wild duck plague virus mutant strains: pMD-335 and pMD-507 diluted 10 times as above were mixed in equal proportions according to the same dilution ratio as templates for PCR amplification, PCR detection was performed according to the reaction system and parameters of Example 1, and the obtained PCR products were analyzed by 1% agarose gel electrophoresis. The results of the sensitivity experiment are shown in Figure 6 .

[0067] Depend on Figures 4 to 6 It can be seen that the numbers from left to right in each figure are 5×10 7 , 5×10 6 , 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 and 5×10-1 The results showed that dual PCR could detect 10 copies of duck plague virus vaccine strain DNA, 10 copies of duck plague virus classic wild strain DNA and 10 copies of duck plague virus variant wild strain DNA.

[0068] Experimental Example 4 Clinical Trial

[0069] Thirty clinically ill duck samples collected from some areas of Shandong from 2019 to 2024 were tested for duck plague virus in the samples according to the detection method provided in Example 1, and the positive samples were confirmed by virus isolation.

[0070] The test results showed that 2 duck plague virus vaccine strains were detected; 5 duck plague virus classic wild strains were detected, mainly concentrated in 2019-2022; and 8 duck plague virus mutant wild strains were detected, mainly concentrated in 2023-2024. All PCR positive results were consistent with the virus isolation results.

[0071] It can be seen that according to the differences between the wild strain of duck plague virus and the UL2 gene and UL56-LORF5 gene of the vaccine virus, two pairs of specific primer sequences are designed, and the DNA of the sample to be tested is extracted as a template to perform a PCR reaction; according to the PCR results, the classic wild strain of duck plague virus, the variant wild strain and the vaccine strain can be distinguished. The PCR identification method for wild strains of duck plague virus and vaccine strains provided by the present invention has the advantages of being simple, rapid, specific, sensitive and economical, and the result judgment method is simple, suitable for use in scientific research, clinical and grassroots laboratories, and has broad application prospects.

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A dual PCR primer set for distinguishing wild strains and vaccine strains of duck plague virus, characterized in that: The dual PCR primer set includes: Primer pair P1 and P2 designed based on the conserved region within the 1002 bp fragment of the duck plague virus UL2 gene; Primer pair P3 and P4 were designed based on the internal conserved region of the 1930 bp fragment of the duck plague virus UL56-LORF5 gene.

2. The dual PCR primer set according to claim 1, characterized in that: The gene sequence of P1 is shown in SEQ ID NO.1; The gene sequence of P2 is shown in SEQ ID NO.2; The gene sequence of P3 is shown in SEQ ID NO.3; The gene sequence of P4 is shown in SEQ ID NO.

4.

3. Use of the dual PCR primer set according to claim 1 or 2 in the preparation of a product for identifying wild-type and vaccine strains of duck plague virus.

4. A kit for distinguishing wild strains and vaccine strains of duck plague virus, characterized in that: It comprises the dual PCR primer set according to claim 1 or 2 and conventional PCR reagents.

5. A dual PCR detection method for distinguishing wild strains and vaccine strains of duck plague virus, characterized in that: The following steps are involved: Extract genomic DNA of the sample to be tested, perform dual PCR amplification using the dual PCR primer set described in claim 1 or 2 or the dual PCR primer set in the kit described in claim 4, and identify the type of the sample based on the amplification result.

6. The dual PCR detection method according to claim 5, characterized in that: The sample types include: one or more of the classical wild strains, variant wild strains and vaccine strains of duck plague virus.

7. The dual PCR detection method according to claim 5, characterized in that: When the PCR amplification band obtained by using primer pair P1 and P2 is 335 bp, and no band is amplified by primer pair P3 and P4, the sample to be tested contains duck plague vaccine strain; When the PCR amplification band obtained by using primer pair P1 and P2 is 862 bp, and the PCR amplification band obtained by using primer pair P3 and P4 is 507 bp, the sample to be tested contains the duck plague classic wild strain; When the PCR amplification band obtained by using primer pair P1 and P2 is 335 bp, and the PCR amplification band obtained by using primer pair P3 and P4 is 507 bp, the sample to be tested contains a mutant wild strain of duck plague.

8. The dual PCR detection method according to claim 5, characterized in that: The reaction system of the double PCR amplification is 25 μL, including: 12.5 μL of 2×PCR Mix, 1.0 μL each of P1, P2, P3 and P4, 2.0 μL of genomic DNA of the sample to be tested and the balance of ddH2O water.

9. The dual PCR detection method according to claim 5 or 8, characterized in that: The reaction procedure of the double PCR amplification includes: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and 35 cycles; extension at 72°C for 7 minutes, and storage at 4°C.

10. Use of the dual PCR primer set according to claim 1 or 2, the kit according to claim 4, or the dual PCR detection method according to any one of claims 5 to 9 in distinguishing wild strains and vaccine strains of duck plague virus.

Images