Multiplex PCR detection kit for duck plague virus, duck tembusu virus and duck rimer's bacillus and application thereof

Abstract

This invention discloses a primer set for multiplex PCR detection of duck plague virus (DTMUV), duck Tembusu virus (DPV), and Riemerella anatipestifer, a kit containing this primer set, and their applications. This invention optimizes and establishes a multiplex PCR detection method for DTMUV, DPV, and Riemerella anatipestifer. Specificity results show that the multiplex PCR detection method established in this invention can only amplify specific bands for DTMUV, DPV, and RA, while no amplification bands are found in PCR amplification products using other pathogen DNA or cDNA as templates, resulting in negative results. Specificity test results demonstrate that the multiplex PCR detection method established in this invention has good specificity. Sensitivity test results show that the minimum detection limit for DTMUV cDNA is 9.855 pg, for RA DNA is 0.9 pg, and for DPV DNA is 0.66 pg. This invention provides an effective technical means for the diagnosis of duck Tembusu virus disease, duck plague, and Riemerella anatipestifer disease.

Description

Technical Field

[0001] This invention relates to a multiplex PCR detection kit and its application, particularly to a multiplex PCR detection kit for duck plague virus, duck Tembusu virus, and duck Riemerella anatipestifer and its application. This invention belongs to the field of biotechnology. Background Technology

[0002] Duck Tembusu virus (DTMUV) is a novel flavivirus belonging to the genus Flaviviridae in the family Flaviviridae. Its genome is a single-stranded positive-sense RNA, characterized by a severe decrease in egg production in laying ducks and neurological symptoms in ducklings. Duckplague virus (DPV) belongs to the genus Marek's virus in the family Herpesviridae. Its genome is a linear, double-stranded DNA, causing an acute, septicemic, and highly lethal infectious disease in ducks, geese, and other Anseriformes birds. The main clinical signs include fever, hemorrhagic diarrhea, and swelling of the head and neck in some affected ducks. It can also cause persistent infection in infected ducks. Riemerella anatipestifer (RA) is a Gram-negative bacillus that primarily causes fibrinous pericarditis, perihepatitis, and air sacculitis in ducks aged 1-8 weeks, accompanied by significant neurological symptoms. Because there is almost no cross-protection between different blood types of RA, immunization is extremely difficult.

[0003] Clinically, the symptoms and pathological findings of duck Tembusu virus disease, duck plague, and duck Riemerella anatipestifer infection are very similar, often making accurate differential diagnosis impossible. Furthermore, due to the expansion of waterfowl farming, increased stocking density, and low management standards, mixed infections of these three pathogens are relatively common. Therefore, timely and accurate identification of the pathogens is often a crucial prerequisite for developing effective control measures for these three infectious diseases. Multiplex PCR technology can detect multiple pathogen genes in the same system. Besides possessing the advantages of rapid, accurate, and sensitive PCR technology, this method shows even greater advantages in the identification of multiple pathogens and the diagnosis of mixed infections, and can significantly reduce testing costs and time. Currently, there are no reports of multiplex PCR detection methods for these three pathogens.

[0004] This invention designs a multiplex PCR detection method that can be used for the detection of DTMUV, DPV and RA, and can provide technical support for the formulation of comprehensive prevention and control measures for these three infectious diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set for multiplex PCR detection of duck plague virus, duck Tembusu virus and duck plague Riemerella anatipestifer, a kit containing the primer set, and its application.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] A multiplex PCR primer set for detecting duck plague virus, duck Tembusu virus, and duck Riemerella anatipestifer, characterized in that the multiplex PCR primer set contains PCR primers for detecting duck plague virus, duck Tembusu virus, and duck Riemerella anatipestifer, respectively. The PCR primers for detecting duck plague virus consist of primer DPV-S shown in SEQ ID NO.1 and primer DPV-A shown in SEQ ID NO.2; the PCR primers for detecting duck Tembusu virus consist of primer DTMUV-S shown in SEQ ID NO.3 and primer DTMUV-A shown in SEQ ID NO.4; and the PCR primers for detecting duck Riemerella anatipestifer consist of primer RA-S shown in SEQ ID NO.5 and primer RA-A shown in SEQ ID NO.6.

[0008] Furthermore, the present invention also proposes the application of the aforementioned multiplex PCR detection primer set in the preparation of reagents for detecting duck plague virus, duck Tembusu virus, and duck plague Riemerella anatipestifer.

[0009] Preferably, the reagent is a multiplex PCR detection reagent.

[0010] Preferably, in the multiplex PCR detection reagent, the final concentrations of primers DTMUV-S and DTMUV-A are both 1.0 μM, the final concentrations of primers RA-S and RA-A are both 0.5 μM, and the final concentrations of primers DPV-S and DPV-A are both 0.5 μM.

[0011] Furthermore, the present invention also proposes a multiplex PCR detection kit for duck plague virus, duck tembusu virus and duck plague Riemerella anatipestifer, wherein the kit contains the aforementioned multiplex PCR detection primer set.

[0012] Preferably, the kit also contains 2×Taq MasterMix.

[0013] Furthermore, the present invention also proposes the application of the aforementioned multiplex PCR detection kit in the preparation of reagents for detecting duck plague virus, duck Tembusu virus, and duck plague Riemerella anatipestifer.

[0014] Preferably, when the multiplex PCR detection kit is used to detect duck plague virus, duck Tembusu virus and duck plague Riemerella anatipestifer, the total reaction volume of the reaction system is 25 μL, including 12.5 μL of 2×Taq MasterMix, 1.0 μL each of DTMUV-S, DPV-S, RA-S, DTMUV-A, DPV-A and RA-A, 1.5 μL of template DNA, and ddH2O added to a total volume of 25.0 μL.

[0015] Preferably, the multiplex PCR detection kit, when detecting duck plague virus, duck Tembusu virus, and duck plague Riemerella anatipestifer, uses the following amplification conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 46.5℃~63.8℃ for 30 s, extension at 72℃ for 30 s, for 30 cycles; and a final extension at 72℃ for 7 min. The PCR products are observed by 1.5% agarose gel electrophoresis.

[0016] Preferably, the annealing temperature is 57℃~61.6℃.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention proposes a primer set for multiplex PCR detection of duck plague virus (DTMUV), duck Tembusu virus (DPV), and Riemerella anatipestifer, a kit containing this primer set, and their applications. This invention optimizes and establishes a multiplex PCR detection method for DTMUV, DPV, and Riemerella anatipestifer. Specificity results show that the multiplex PCR detection method established in this invention can only amplify specific bands for DTMUV, DPV, and RA, while no amplification bands are found in PCR amplification products using other pathogen DNA or cDNA as templates, resulting in negative results. Specificity test results demonstrate that the multiplex PCR detection method established in this invention has good specificity. Sensitivity test results show that the minimum detection limit for DTMUV cDNA is 9.855 pg, for RA DNA is 0.9 pg, and for DPV DNA is 0.66 pg. The multiplex PCR detection method established in this invention, applicable to the detection of DTMUV, DPV, and RA, can provide an effective technical means for the diagnosis of these three infectious diseases. Attached Figure Description

[0019] Figure 1 This is a single PCR amplification result;

[0020] Among them, M.DNALadder 2000; 1-3. are the individual PCR amplification products of DTMUV, RA and DPV respectively;

[0021] Figure 2 This is the result of multiplex PCR amplification;

[0022] Among them, M.DNA Ladder 2000; 1. DTMUV and RA dual PCR product; 2. DTMUV and DPV dual PCR product; 3. RA and DPV dual PCR product; DTMUV, RA and DPV multiplex PCR product;

[0023] Figure 3 Results of annealing temperature optimization for multiplex PCR;

[0024] Among them, M.DNA Ladder 2000; 1. Annealing temperature 65℃; 2. Annealing temperature 63.8℃; 3. Annealing temperature 61.6℃; 4. Annealing temperature 57℃; 5. Annealing temperature 52.7℃; 6. Annealing temperature 48.9℃; 7. Annealing temperature 46.5℃; 8. Annealing temperature 45℃;

[0025] Figure 4 The results represent the optimal primer concentration.

[0026] Among them, M.DNA Ladder 2000; 1. Group A; 2. Group B; 3. Group C; 4. Group D; 5. Group E; 6. Group F; 7. Group G; 8. Group H; 9. Group I;

[0027] Figure 5 The results represent the optimal primer concentration.

[0028] Among them, M.DNA Ladder 2000; 1. A mixture of cDNA from DTMUV, DNA from DPV, and DNA from RA; 2. Newcastle disease virus cDNA; 3. Duck parvovirus DNA; 4. Duck-derived Escherichia coli DNA; 5. Duck-derived Salmonella DNA; 6. Duck-derived Barmonella DNA; 7. Duck-derived Streptococcus DNA;

[0029] Figure 6 These are the results of a sensitivity experiment;

[0030] Among them, A: M.DNA Ladder 2000; 1.98550pg; 2.9855pg; 3.985.5pg; 4.98.55pg; 5.9.85pg; 6.0.9855pg; 7.ddH2O;

[0031] B: M.DNA Ladder 2000; 1.9000pg; 2.900pg; 3.90pg; 4.9pg; 5.0.9pg; 6.0.09pg; 7.ddH2O;

[0032] C: M.DNA Ladder 2000; 1.6600pg; 2.660pg; 3.66pg; 4.6.6pg; 5.0.66pg; 6.0.09pg; 7.ddH2O. Detailed Implementation

[0033] The present invention will be further illustrated below through experiments and embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0034] Example 1: Establishment of a multiplex PCR detection method for duck plague virus, duck tembusu virus, and duck plague Riemerella anatipestifer.

[0035] 1. Materials and Methods

[0036] 1.1 Virus strains, bacterial strains and pathogenic materials

[0037] The live vaccine for Newcastle disease in chickens (La Sota strain) and the live vaccine for duck plague virus were produced by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.; the live vaccine for duck Tembusu virus (FX2010-180P strain) was produced by Qingdao Yibang Biotechnology Co., Ltd.; duck parvovirus, avian Escherichia coli, Salmonella, Pasteurella multocida, Streptococcus, Riemerella anatipestifer, and pathogenic materials were stored in the Preventive Veterinary Laboratory of the College of Animal Science and Technology, Xinyang Agricultural and Forestry University.

[0038] 1.2 Main Reagents

[0039] The bacterial genomic DNA rapid extraction kit was manufactured by Sangon Biotech (Shanghai) Co., Ltd.; the viral genomic DNA / RNA extraction kit was manufactured by Tiangen Biotech (Beijing) Co., Ltd.; 2×Taq Master Mix and DNA Ladder 2000 were manufactured by Nearshore Protein Technology Co., Ltd.; the RevertAid RT reverse transcription kit was manufactured by Thermo Fisher Scientific; agarose was manufactured by BBI; and all other chemical reagents were of analytical grade.

[0040] 1.3 Primer Design and Synthesis

[0041] Homology comparisons of published DTMUV NS5, DPV US4, and RAOMPA gene sequences in GenBank were performed using DNAMAN7 software (see Table 1 for reference sequence numbers). Conserved regions were selected, and primers for detecting the three pathogens were designed using Primer Premier 5.0. The three pathogens could be distinguished based on the size difference of the PCR products. Primer sequences are shown in Table 2 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0042] Table 1 Reference Serial Numbers

[0043]

[0044] Table 2 Primer sequences

[0045]

[0046] 1.4 Nucleic acid extraction and cDNA synthesis

[0047] DNA was extracted from tissue samples and bacteria using a bacterial genomic DNA rapid extraction kit. DNA or RNA was extracted from tissue samples or viruses using a viral genomic DNA / RNA extraction kit. The specific procedures were performed according to the manufacturer's instructions. The extracted RNA was used to synthesize cDNA according to the RevertAid RT reverse transcription kit instructions. The reaction mixture consisted of 19 μL RNA, 2.0 μL dNTP Mix (10 mM), 6.0 μL 5× Reaction Buffer, 1.5 μL Random Primer, 0.75 μL RevertAid Reverse Transcriptase, and 0.75 μL RiboLock RNase Inhibitor. The reaction conditions were: 25℃ for 5 min, 42℃ for 60 min, 70℃ for 5 min, and stored at -20℃ for later use.

[0048] 1.5 Single PCR

[0049] The total reaction volume was 25 μL, including 12.5 μL of 2×Taq Master Mix, 1.0 μL of DTMUV-S (or DPV-S or RA-S, 20 μM), 1.0 μL of DTMUV-A (or DPV-A or RA-A, 20 μM), 1.5 μL of DNA, and ddH2O added to a total volume of 25.0 μL. Amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles; and a final extension at 72℃ for 7 min. PCR products were observed by 1.5% agarose gel electrophoresis. PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for nucleotide sequencing.

[0050] 1.6 Multiplex PCR

[0051] The reaction system and amplification conditions are shown in 1.5, except that the primers are changed to 1.0 μL each of DTMUV-S, DPV-S, DTMUV-A and DPV-A, or 1.0 μL each of DTMUV-S, RA-S, DTMUV-A and RA-A, and 1.0 μL each of DPV-S, DPV-A, RA-S and RA-A, or 1.0 μL each of DTMUV-S, DPV-S, RA-S, DTMUV-A, DPV-A and RA-A.

[0052] 1.7 Optimization of Multiplex PCR Amplification Conditions

[0053] 1.7.1 Optimal Annealing Temperature Optimization

[0054] The DTMUV, DPV, and RA triple PCR reaction system and amplification program were performed according to section 1.6, with annealing temperatures set at 65℃, 63.8℃, 61.6℃, 57℃, 52.7℃, 48.9℃, 46.2℃, and 45℃, respectively. The optimal annealing temperature for multiplex PCR was determined by agarose gel electrophoresis.

[0055] 1.7.2 Optimal Primer Concentration Optimization

[0056] The triple PCR reaction system and amplification procedure for DTMUV, DPV, and RA were performed according to section 1.6. The final concentration combinations of primers for the PCR amplification of the three pathogens are shown in the table.

[0057] Table 3 Primer final concentration combinations

[0058]

[0059]

[0060] 1.8 Specificity test

[0061] Genomic DNA and RNA were extracted from Newcastle disease virus (NDV) in chickens, duck parvovirus, duck-derived Escherichia coli, Salmonella, Pasteurella multocida, and Streptococcus according to procedure 1.4, and the RNA was reverse transcribed into cDNA. Using cDNA as a template, PCR detection was performed according to the optimal reaction system and amplification conditions determined in steps 1.6 and 1.7. A mixture of DNA from DTMUV, DPV, and RA was used as a control.

[0062] 1.9 Sensitivity Experiment

[0063] The nucleic acid concentrations of DTMUV, DPV, and RA were determined using an ultra-micro UV spectrophotometer, and then serially diluted 10-fold (totaling 6 dilutions). Using these as templates, PCR detection was performed according to the optimal reaction system and amplification conditions determined in sections 1.6 and 1.7, with ddH2O used as a negative control.

[0064] 1.10 Clinical Sample Testing

[0065] The multiplex PCR detection method established in this invention was used to detect 36 clinically collected samples. At the same time, three single PCR methods for pathogens were used for detection, and the concordance rate between the multiplex PCR and single PCR detection results was compared.

[0066] 2 Results

[0067] 2.1 Results of single-item PCR amplification

[0068] The results of single-sample PCR detection of DTMUV, DPV, and RA are shown in Figure 1. Using cDNA extracted from DTMUV genomic RNA as a template and DTMUV-S / DTMUV-A primers as primers, the PCR product was a specific band of approximately 220 bp. Using RA DNA as a template and RA-S / RA-A primers as primers, the PCR product was a specific band of approximately 308 bp. Using DPV DNA as a template and DPV-S / DPV-A primers as primers, the PCR amplification product was a band of approximately 551 bp. All these results were consistent with expectations. Compared with the DL2000 Marker, the sizes of the PCR amplification products of the three pathogens were clearly distinguishable. Nucleotide sequencing confirmed that the three PCR products were gene fragments of DTMUV, RA, and DPV, respectively. 2.1 Multiplex PCR Amplification Results

[0069] Multiplex PCR test results as follows Figure 2 As shown, using a mixture of DTMUV cDNA and RA DNA as templates, the PCR products showed bands of 220bp and 308bp, respectively; using a mixture of DTMUV cDNA and DPV DNA as templates, the PCR products showed bands of 220bp and 551bp, respectively; using a mixture of DPV and RA DNA as templates, the PCR products showed bands of 551bp and 308bp, respectively; using a mixture of DTMUV cDNA, DPV DNA, and RA DNA as templates, the PCR products showed bands of 220bp, 551bp, and 308bp, respectively. All these results were consistent with expectations. Using the DL2000 Marker as a reference, the three pathogens could be clearly distinguished based on the size of their PCR products.

[0070] 3.3 Optimization results of annealing temperature for multiplex PCR amplification

[0071] The optimal annealing temperature optimization results for the multiplex PCR detection method established in this invention are as follows: Figure 3 As shown, when the annealing temperature is between 46.5℃ and 63.8℃, the multiplex PCR detection method can simultaneously amplify 551bp, 308bp, and 220bp bands, thus simultaneously detecting three pathogens. Among them, when the annealing temperature is 61.6℃ and 57℃, the PCR product bands of the three pathogens are relatively obvious. Therefore, the optimal annealing temperature range for the multiplex PCR detection method established in this invention is between 57℃ and 61.6℃.

[0072] 3.4 Results of Optimal Primer Concentration Optimization

[0073] The optimization results of the concentration of three primer pairs in the multiplex PCR detection method established in this invention are as follows: Figure 4As shown, all nine primer concentration combinations amplified bands of 551 bp, 308 bp, and 220 bp, respectively. Among them, the PCR amplification products of the three primer concentration combinations in group E were the clearest: primers DTMUV-S and DTMUV-A were both at a final concentration of 1.0 μM, primers RA-S and RA-A were both at a final concentration of 0.5 μM, and primers DPV-S and DPV-A were both at a final concentration of 0.5 μM.

[0074] 3.5 Specificity test results

[0075] The specificity test results of the multiplex PCR detection method established in this invention are as follows: Figure 5 As shown, using a mixture of DTMUV cDNA, DPV, and RA DNA as templates, the PCR amplification products showed bands of 551 bp, 308 bp, and 220 bp, while no amplification bands were found in the PCR amplification products using other pathogen DNA or cDNA as templates, and the results were judged as negative. Specificity test results demonstrate that the multiplex PCR detection method established in this invention has good specificity.

[0076] 3.6 Sensitivity Test Results

[0077] The cDNA concentration of DTMUV was 0.657 μg / μL, while the DNA concentrations of RA and DPV were 0.06 μg / μL and 0.044 μg / μL, respectively. Sensitivity test results are as follows... Figure 6 As shown, the minimum detection limit for DTMUV cDNA established by this invention is 9.855 pg, the minimum detection limit for RA DNA is 0.9 pg, and the minimum detection limit for DPV DNA is 0.66 pg.

[0078] 3.7 Preliminary Clinical Application Results

[0079] Fifty-four clinically collected duck samples were tested using single PCR detection methods for RA, DTMUV, and DPV. The results are shown in Table 4. There were 20 cases of simple RA, 12 cases of DTMUV, and 4 cases of DPV infection; 13 cases of mixed RA and DTMUV infection; 2 cases of mixed RA and DPV infection; and 3 cases where none of the three pathogens were detected. In comparison, the multiplex PCR detection method established in this invention showed that in one case of mixed RA and DTMUV infection, DTMUV was not detected, and the case was determined to be a simple RA infection. All other detection results were consistent with the single PCR detection results. These results indicate that the multiplex PCR detection method established in this invention has a concordance rate of 98.1% with the single PCR detection method.

[0080] Table 4. Statistics of PCR test results of clinically collected pathological materials

[0081]

Claims

1. A set of primers for multiplex PCR detection of duck plague virus (DPV), duck Tembusu virus (DTMUV), and Riemerella anatipestifer (RA), characterized in that, The multiplex PCR detection primer set contains PCR primers for detecting duck plague virus, duck Tembusu virus, and duck Riemerella anatipestifer, respectively. The PCR primers for detecting duck plague virus consist of primer DPV-S shown in SEQ ID NO.1 and primer DPV-A shown in SEQ ID NO.

2. The PCR primers for detecting duck Tembusu virus consist of primer DTMUV-S shown in SEQ ID NO.3 and primer DTMUV-A shown in SEQ ID NO.

4. The PCR primers for detecting duck Riemerella anatipestifer consist of primer RA-S shown in SEQ ID NO.5 and primer RA-A shown in SEQ ID NO.

6.

2. The application of the multiplex PCR detection primer set according to claim 1 in the preparation of reagents for detecting duck plague virus, duck Tembusu virus and duck plague Riemerella anatipestifer.

3. The application as described in claim 2, characterized in that, The reagent described is a multiplex PCR detection reagent.

4. The application as described in claim 3, characterized in that, In the multiplex PCR detection reagent, the final concentrations of primers DTMUV-S and DTMUV-A are both 1.0 μM, the final concentrations of primers RA-S and RA-A are both 0.5 μM, and the final concentrations of primers DPV-S and DPV-A are both 0.5 μM.

5. A multiplex PCR detection kit for duck plague virus, duck Tembusu virus, and duck plague Riemerella anatipestifer, characterized in that, The kit contains the multiplex PCR detection primer set as described in claim 1.

6. The multiplex PCR detection kit as described in claim 5, characterized in that, The kit also contains 2×TaqMasterMix.

7. The use of the multiplex PCR detection kit according to claim 5 or 6 in the preparation of reagents for detecting duck plague virus, duck Tembusu virus and duck plague Riemerella anatipestifer.

8. The application as described in claim 7, characterized in that, The multiplex PCR detection kit described above, when detecting duck plague virus, duck Tembusu virus and duck plague Riemerella anatipestifer, has a total reaction volume of 25 μL, including 12.5 μL of 2×TaqMasterMix, 1.0 μL each of DTMUV-S, DPV-S, RA-S, DTMUV-A, DPV-A and RA-A, 1.5 μL of template DNA, and ddH2O added to a total volume of 25.0 μL.

9. The application as described in claim 8, characterized in that, The multiplex PCR detection kit described above, when detecting duck plague virus, duck Tembusu virus, and duck plague Riemerella anatipestifer, uses the following amplification conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 46.5℃~63.8℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles; and 72℃ final extension for 7 min. The PCR products are observed by 1.5% agarose gel electrophoresis.

10. The application as described in claim 9, characterized in that, The annealing temperature is 57℃~61.6℃.

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