SYBR Green I fluorescent quantitative PCR detection primer for detecting MDGPV virulent virus, MDGPV attenuated virus and MDPV and application of SYBR Green I fluorescent quantitative PCR detection primer

Abstract

The invention relates to SYBR Green I real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection primers for simultaneously identifying and detecting MDGPV virulent virus, MDGPV attenuated virus and MDPV and application of the SYBR Green I real-time fluorescent quantitative PCR detection primers. The sequences of the primers are shown as SEQ ID NO.1-2. The application of the primers in preparation of a kit for simultaneously identifying and detecting the MDGPV virulent virus, the MDGPV attenuated virus and the MDPV. The detection method established by using the primer can simultaneously detect MDGPV virulent virus, MDGPV attenuated virus and MDPV, has good specificity, has no cross reaction with other main waterfowl source viruses, and has strong specificity; the lower limit of detection on a pMD-MDGPVPT-ITR plasmid standard substance, a pMD-MDGPVD-ITR plasmid standard substance and a pMD-MDPV-ITR plasmid standard substance is 1.0 * 10 < 1 > copy / mu L; according to the method, the variation coefficients of intra-group and inter-group repeatability tests are both less than 1.0%, and the repeatability is good.

Description

Technical Field

[0001] The invention relates to a SYBR Green I fluorescent quantitative PCR detection primer for simultaneously identifying and detecting strong MDGPV, weak MDGPV and MDPV and an application thereof. Background Art

[0002] The morbidity of Muscovy duck parvovirus disease (commonly known as the "three-week disease" of Muscovy ducks) is 27% to 60%, and the mortality rate is 22% to 60%. Most of the ducks that have recovered from the disease become dead ducks. The pathogen is Muscovy duck parvovirus (MDPV). In Muscovy duck breeding areas, it was found that the young Muscovy ducks often had diarrhea of ​​varying degrees, and some sick ducks had intestinal mucosal shedding and embolism. The morbidity of the disease was 50% to 70%, and the mortality rate was 40% to 65%. The pathogen was a new chimeric strain produced by the natural recombination of classical goose parvovirus (C-GPV) and MDPV, namely Muscovy duck origin-goose parvovirus (MDGPV). Both of these infectious diseases are important diseases that seriously affect the Muscovy duck farming industry in my country. In clinical practice, the two are often mixed infections, causing great economic losses to the Muscovy duck farming industry. MDGPV belongs to the Anseriform dependoparvovirus 1 species of the Dependovirus genus of the Parvoviridae family. It has certain antigenic cross-reactivity with the same species of MDPV, and these two viruses can also naturally infect Muscovy ducks. Therefore, it is not easy to distinguish MDGPV from MDPV in polyclonal antibody immunological detection. In recent years, MDGPV has been constantly mutating. The recombination and mutation between different virus strains have made the clinical manifestations of Muscovy duck gosling plague more complicated. The pathological changes in pathological autopsy are often different from those of typical Muscovy duck gosling plague, which has caused great difficulties for veterinary clinical diagnosis. At present, the attenuated MDGPV vaccine used in Muscovy duck breeding areas in my country has the characteristics of good safety, strong immunogenicity, long duration of immune protection and high immune protection efficiency, which plays a very important role in the prevention and control of Muscovy duck gosling plague. However, the application of MDGPV attenuated vaccine makes the clinical differential diagnosis of attenuated and virulent MDGPV cases complicated. In order to facilitate the purification of MDGPV and MDPV in Muscovy duck farms, it is particularly important to establish a sensitive, specific, and reproducible multiplex PCR detection method for rapid virological differentiation between strong virus infection and weak virus inoculation.

[0003] Therefore, in order to establish a rapid identification and detection method for the virulent and attenuated MDXPV and MDPV, this study designed a pair of specific primers based on the difference sequences of the 3'-ITR genes of the virulent parent strain of MDXPV and the vaccine strain and representative strain of MDPV. Only a pair of specific fluorescent quantitative polymerase chain reaction (qPCR) primers was needed to successfully establish a SYBR Green IqPCR identification and detection method for rapidly distinguishing the virulent MDXPV, attenuated MDXPV and MDPV, which provides effective technical support for the clinical real-time monitoring of the prevalence of MDPV and MDXPV in Muscovy duck flocks and immune prevention and control. Summary of the invention

[0004] The purpose of the present invention is to provide a SYBRGreen I qPCR detection primer and application for simultaneously identifying and detecting MDDPV virulent, MDDPV weakly virulent and MDPV, and to establish a SYBR Green I qPCR detection method for simultaneously identifying and detecting MDDPV virulent, MDDPV weakly virulent and MDPV using the primer.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A SYBR Green I qPCR detection primer for simultaneously identifying and detecting MDGPV virulent, MDGPV weakly virulent, and MDPV, the sequence of the primer is as follows:

[0007] WPV-ITR-F: 5'-GCATGCGCCCGATCTGCCATGAA-3' (SEQ ID NO. 1), WPV-ITR-R: 5'-GTGCTTCCCGGTCATGTGTTTCC-3' (SEQ ID NO. 2).

[0008] The SYBR Green I qPCR detection primers are used in preparing a kit for simultaneously identifying and detecting MDGPV virulent strains, MDGPV mild strains and MDPV.

[0009] The present invention also provides a kit for simultaneously identifying and detecting the highly toxic MDGPV, the weakly toxic MDGPV and the MDPV, wherein the kit comprises the SYBR Green I qPCR detection primer.

[0010] The present invention also provides a SYBR Green I fluorescent quantitative PCR detection method for simultaneously identifying and detecting MDGPV virulent, MDGPV virulent and MDPV using the SYBR Green I qPCR detection primers, and the method is not for the purpose of diagnosis, and the PCR reaction system thereof is: the following components are contained in a 20 μL system: Top Green qPCRSuperMix 10μL, 0.5μL of primers WPV-ITR-F and WPV-ITR-R at a concentration of 10μmol / L, 1μL of positive templates corresponding to the three viruses of MDGPV, MDGPV and MDPV, or 3μL of template DNA of the sample to be tested, and make up to 20μL with Nuclease-free Water;

[0011] The PCR amplification conditions were: 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, for 40 cycles, while collecting the fluorescence signal and making the corresponding melting curve.

[0012] Compared with the prior art, the advantages of the present invention are:

[0013] According to the difference in the highly conservative 3'-ITR gene nucleotide sequence between a strong strain of Muscovy duck-origin goose parvovirus (MDGPV), a weak strain of MDXPV and a representative strain of Muscovy duck parvovirus (MDPV), a pair of specific fluorescent quantitative primers are designed, and after optimizing the reaction conditions, the recombinant plasmids pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR of the 3'-ITR genes of the strong PT strain of MDXPV, the weak D strain of MDXPV and the MDPV-P strain are used as plasmid standards to establish a SYBR Green I qPCR method for distinguishing and detecting strong MDGPV, weak MDGPV and MDPV. The results showed that the method could detect MDGPV virulent, MDGPV attenuated and MDPV simultaneously with good specificity and no cross reaction with other major waterfowl viruses. The detection limits of pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR plasmid standards were all 1.0×10 1 The coefficient of variation of the intra-group and inter-group repeatability tests of this method was less than 1.0%, and the repeatability was good.

[0014] The method was used to detect 35 tissue samples of clinically ill Muscovy ducks. The positive rates of MDGPV and MDPV were 11.43% and 8.75%, respectively, and the mixed rate was 8.75%. No MDGPV was detected. The results of the SYBR Green I fluorescence quantitative PCR method for clinical samples were consistent with the results of sequencing analysis.

[0015] The SYBR Green I qPCR method established in this study provides new technical support for the rapid identification of virulent MDDPV, attenuated MDDPV and MDPV from the perspective of etiology, and provides a new method for the purification of Muscovy duck "three-week disease" and Muscovy duck gosling plague in large-scale Muscovy duck farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Standard curves of the SYBR Green I qPCR method, where A: MDGPV-PT standard curve; B: MDGPV-D standard curve; C: MDPV-P standard curve.

[0017] Figure 2 SYBR Green I qPCR melting curve, where 1: MDGPV-D melting temperature is approximately 83.8±0.5℃; 2: MDGPV-PT melting temperature is approximately 85.2±0.5℃; 3: MDPV-P melting temperature is approximately 86.1±0.5℃.

[0018] Figure 3 SYBR Green I qPCR specificity test results, where 1: MDGPV-D positive plasmid; 2: MDPV-P positive plasmid; 3: MDGPV-PT positive plasmid; 4: MDGPV-D positive nucleic acid; 5: MDPV-P positive nucleic acid; 6: MDGPV-PT positive nucleic acid; 7-15: other viruses; 16: negative control.

[0019] Figure 4 MDGPV-PT sensitivity test, where 1-9: 1×10 8 -1×10 0 Copies / μLMDGPV-PT positive plasmid, 10: negative control.

[0020] Figure 5 MDGPV-D sensitivity test results, 1-9: 1×10 8 -1×10 0 Copies / μLMDGPV-D positive plasmid, 10: negative control.

[0021] Figure 6 is the sensitivity test result of MDPV-P, where 1-9: 1×10 8 -1×100 Copies / μLMDPV-P positive plasmid, 10: negative control. DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0023] 1 Materials and methods

[0024] 1.1 Main experimental materials

[0025] Muscovy duck reovirus (MDRV), novel duck reovirus (NDRV), duck viral hepatitis virus type I (DHV-I), duck paramyxovirus (DPMV), duck plague virus (DEV), duck Tembusu virus (DTMUV), duck short-beaked dwarf syndrome virus (SBDS-GPV), classical goose parvovirus (C-GPV), Muscovy duck parvovirus (MDPV) standard virus P strain, Muscovy duck gosling plague virus (MDGPV) virulent PT strain, Muscovy duck gosling plague virus (MDGPV) attenuated D strain are preserved in the Animal Virus Laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0026] The pMD-18T plasmid and Escherichia coli competent DH5α were purchased from Takara Biotechnology (Dalian) Co., Ltd. Simple Viral DNA / RNAKit(ER211-01), Top Green qPCR SuperMix (AQ131-01) was purchased from Beijing Quanshijin Biotechnology Co., Ltd. 2×Rapid Taq Master Mix (P222-01), FastPure Plasmid Mini Kit (DC201-01), and FastPure Gel DNA Extraction Mini Kit (DC301-01) were purchased from Novozyme Biotechnology Co., Ltd. FastKing one-step genomic cDNA first-strand synthesis premix (KR118) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The 96 fluorescence quantitative PCR instrument was purchased from Roche, USA.

[0027] 1.2 Primer design

[0028] The present invention selects the 3'-ITR genes of the strong and weak strains of MDXPV, the weak and D strains of MDXPV and the standard strain of MDPV as target genes, selects the nucleotide regions that can represent the strong and weak strains of MDXPV and the sequence characteristics of the ITR genes of each MDPV through BLAST comparison, designs multiple primers using Oligo 6.0 software, and finally successfully screens a pair of SYBR GreenI qPCR primers (WPV-ITR-F / WPV-ITR-R) through optimization and screening, and uses the pair of primers to construct plasmid standards and establish a detection method. The primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific information is shown in Table 1.

[0029] Table 1 Virus nucleic acid detection primer information

[0030]

[0031] 1.3 Extraction of viral genes

[0032] Using a viral DNA / RNA genome extraction kit, MDRV, NDRV, DHV-I, DPMV, and DTMUV were extracted and reverse transcribed into cDNA, and DEV, SBDS-GPV, and C-GPV genomic DNA were extracted and stored at -80°C for future use.

[0033] 1.4 Preparation of plasmid standards

[0034] The genomic DNA of the virulent PT strain of MDXPV, the attenuated D strain of MDXPV and the standard P strain of MDPV were used as templates, and the primers in Table 1 were used to amplify the ITR of MDXPV-PT, MDXPV-D and MDPV-P, respectively. PCR reaction system (25 μL): 2×RapidTaqMasterMix 12.5 μL, 1 μL of each of the 10 μmol / L upstream and downstream primers, 2 μL of template, and 8.5 μL of ddH2O. PCR reaction conditions: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s, for 33 cycles; 72℃ final extension for 5 min. The target PCR product was recovered using a gel recovery kit, connected to the pMD-18T simple vector, and transformed into the competent E. coli DH5α, placed in a 37°C shaker for bacterial growth for 1 hour, and the bacterial solution was spread on LB solid medium containing ampicillin for overnight culture. After that, a single colony on the plate was selected for purification culture. After PCR identification, the plasmid was extracted. The positive bacterial solution was sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing. The correctly sequenced recombinant plasmid was used as a positive control standard.

[0035] 1.5 Optimization of SYBR Green I qPCR reaction system and conditions

[0036] Using the positive standards of MDGPV-PT, MDGPV-D and MDPV-P strains as templates, a 20 μL system was used: Top Green qPCR SuperMix 10μL, WPV-ITR upstream and downstream primers (10μmol / L) 1μL each, positive template 1μL each, supplemented to 20'μL with Nuclease-free Water. Nine annealing temperatures, including 55℃, 56℃, 57.2℃, 58.5℃, 59.2℃, and 60℃, were set for SYBR Green I fluorescent quantitative PCR of MDPV-P, MDGPV-PT, and MDGPV-D virus strains. According to the optimized optimal annealing temperature, the primers with a concentration of 10μmol / L were optimized in the volume range of 0.5-2.5μL and the templates in the volume range of 0.5-2.5μL by matrix method.

[0037] 1.6 Drawing of standard curve and melting curve

[0038] The plasmid standards of MDGPV-PT, MDGPV-D and MDPV-P strains were diluted 10-fold, and 1×10 6 Copies / μL~1×10 0 The plasmid with 7 dilutions of copies / μL was used as the template, and 3 replicates were set for each dilution, and a negative control group was set up for amplification using the SYBR Green IqPCR method optimized in step 1.5. The quantitative standard curve was drawn with the number of copies of the initial template as the horizontal axis and the Ct value of the cycle as the vertical axis, and a melting curve was generated at the same time.

[0039] 1.7 Specificity test

[0040] Using DNA of MDPV-P, MDGPV-PT, MDGPV-D, C-GPVNP5, SBDS-GPV M15, DEV-fj1, and cDNA of MDRVMW9710, NDRVNP03, DHV-I NA, and DPMV LG as templates, a mixture of three recombinant plasmid standards as a positive control, and Nuclease-free Water as a negative control, each sample was repeated three times and amplified using the established SYBR Green I qPCR method to evaluate the specificity of this method.

[0041] 1.8 Sensitivity test

[0042] The plasmid standards of MDGPV-PT, MDGPV-D and MDPV-P strains were diluted 10-fold, and 1×10 8 ~1×10 0A total of 9 dilutions of plasmids with different copies / μL were used as templates (three replicates were set) and the detection was performed according to the optimized SYBR Green I qPCR reaction conditions to verify the sensitivity of the method.

[0043] 1.9 Repeatability test

[0044] Select 1×10 of the plasmid standards of MDGPV-PT, MDGPV-D and MDPV-P strains 5 , 1×10 4 , 1×10 3 Three dilutions of 100 copies / μL were used as templates, and three replicates were set for each concentration. The optimized SYBR Green I qPCR method was used to perform intra-batch and inter-batch tests on three reaction systems of the same batch and three reaction systems of different batches, and the intra-batch and inter-batch coefficients of variation were calculated to evaluate the repeatability of the method. A negative control was also set up.

[0045] 1.10 Clinical sample testing

[0046] 35 samples of liver, spleen, kidney and small intestine tissues of clinically ill Muscovy ducks were cut into small pieces weighing about 15 g and placed in a 2 mL sterile centrifuge tube. An appropriate amount of PBS was added and the samples were frozen and thawed three times. The supernatant was extracted after grinding and centrifugation with a tissue grinder and total DNA was extracted and tested using the optimized SYBR Green I qPCR. The amplified positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence determination, and the consistency rate between the PCR method and the sequencing results was calculated to verify the accuracy of the established method.

[0047] 2 Results

[0048] 2.1 Identification results of recombinant plasmid standards

[0049] The total DNA of MDXPV-PT, MDXPV-D and MDPV-P strains was extracted as templates, and the corresponding primers were used for PCR amplification to obtain the target fragments of 3'-ITR genes of MDXPV-PT, MDXPV-D and MDPV-P strains, which were all 100bp, consistent with the expected results. The target fragments were cloned into pMD18-T to construct template plasmids pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR, and sequenced after PCR identification. The sequencing results were compared with the viral gene sequences of MDXPV-PT, MDXPV-D and MDPV-P published by NCBI, and the homology was 100%, indicating that the positive standard plasmid was successfully constructed. The concentrations of the three recombinant plasmids were determined by ultraviolet spectrophotometer and converted into copy numbers (copies / μL) according to the formula. The results showed that the concentrations of pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR were 3.5×10 9 copies / μL, 2.4×10 9 copies / μL and 4.5×10 9 Copies / μL. All three were diluted to 1.0×10 9 copies / μL, as recombinant plasmid standard, stored at -20℃.

[0050] 2.2 Determination of the optimal reaction conditions for SYBR Green I qPCR

[0051] The results of optimization of each reaction condition by matrix method showed that the best reaction system of SYBR Green I qPCR was 20μL: 2×TransStartTop Green qPCR SuperMix 10μL, 10μmol / L upstream and downstream primers 0.5μL each, template was a mixture of recombinant plasmid standards pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR in equal proportions 3μL, Nuclease-free Water 6μL. Amplification reaction program: 94℃30s; 94℃5s, 60℃30s, 40 cycles, and fluorescence signal was collected at the same time.

[0052] 2.3 Establishment of qPCR standard curve and melting curve

[0053] According to the optimized SYBR Green I qPCR reaction conditions, 10 1 ~10 7 dilution, i.e. containing 1×10 6 Copies / μL~1×10 0 Copies / μLpMD-MDGPVPT-ITR, 1×106 Copies / μL~1×10 0 copies / μLpMD-MDGPVD-ITR and 1×10 6 Copies / μL~1×10 0 The standard curve and melting curve of SYBR Green I qPCR were obtained by using pMD-MDPV-ITR as template. The linear relationship between the logarithm (X) of the copy number of MDGPV-PT, MDGPV-D, and MDPV-P and the cycle threshold (Ct) was Y = -3.2455X + 38.982, and the correlation coefficient R 2 =0.9994; Y = -3.4898X + 39.423, correlation coefficient R 2 =0.9996; Y = -3.3355X + 38.123, correlation coefficient R 2 =0.9985. The correlation coefficient of the three standard curves (R 2 ) were all above 0.998. This indicates that the three plasmid standards are 6 Copies / μL~1×10 0 There was a good linear relationship between copies / μL and their respective Ct values ​​( Figure 1 ). The melting temperatures of MDGPV-PT, MDGPV-D, and MDPV-P were approximately 85.2±0.5℃, 83.8±0.5℃, and 86.1±0.5℃, respectively. No melting point peak appeared in the negative control, while the standard amplification showed a single peak, indicating that there was no contamination in this experiment and the data obtained were highly reliable ( Figure 2 ).

[0054] 2.4 Specificity test results

[0055] The DNA of MDGPV-PT, MDGPV-D, MDPV-P, DD-GPVNP5, SBDS-GPV M15, DEV-fj1, and cDNA of MDRVMW9710, NDRVNP03, DHV-INA, and DPMV LG were used as templates for amplification using the established SYBR Green I qPCR. The results showed that the recombinant plasmid standards pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR, and pMD-MDPV-ITR as positive controls, and the DNA of MDGPV-PT, MDGPV-D, and MDPV-P isolated viruses all showed amplification curves, while the DNA or cDNA of other viruses and the negative controls showed no amplification curves ( Figure 3 ). This indicates that the established SYBR Green IqPCR method has strong specificity.

[0056] 2.5 Sensitivity test results

[0057] The optimized SYBR Green I qPCR method was used to detect serially diluted pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR positive plasmid standard templates (1×10 8 -1×10 1 The results showed that a good "S"-shaped amplification curve was obtained, and the minimum detection limit was 1×10 1 copies / μL, demonstrating that this method has good sensitivity.

[0058] 2.6 Repeatability test results

[0059] The established method was used to analyze three serial dilutions (1×10 5 , 1×10 4 With 1×10 3 The intra-assay and inter-assay repeatability tests were performed on the recombinant plasmid standards pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPVP-ITR of MDXPV-PT, MDXPV-D and MDPV-P (1000 copies / μL). The results showed that the intra-assay and inter-assay coefficients of variation of MDXPV-PT were 0.14%-0.26% and 0.40%-0.80%, respectively; the intra-assay and inter-assay coefficients of variation of MDXPV-D were 0.12%-0.28% and 0.41%-0.67%, respectively; the intra-assay and inter-assay coefficients of variation of MDPV-P were 0.14%-0.28% and 0.45%-0.60%, respectively (Table 1). The intra-assay and inter-assay coefficients of variation of the three groups of plasmids were all lower than 1%, indicating that the SYBR Green I qPCR method established in this study had good stability.

[0060] Table 2 Repeatability results of SYBR Green I fluorescence quantitative PCR detection

[0061]

[0062] 2.7 Clinical sample testing

[0063] The SYBR Green I qPCR method established in this study was used to detect 35 tissue samples of clinically ill ducks. The results showed that the detection rate of strong MDGPV was 11.43% (4 / 35), the detection rate of MDPV was 8.75% (3 / 35), the mixed infection rate of strong MDGPV and MDPV was 8.75% (3 / 35), and no weak MDGPV was detected. The positive samples were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the sequence was compared using BLAST online software. The results showed that the SYBR Green I qPCR method established in this study had a 100% consistency with the sequencing results, indicating that the SYBRGreen I qPCR method established in this study has the characteristics of high sensitivity and strong stability, and is suitable for the detection of clinical samples.

[0064] 3 Discussions

[0065] The "three-week disease" of Muscovy ducks and the plague of Muscovy ducks and goslings are both highly contagious viral infectious diseases with high mortality rates that seriously threaten the healthy development of the waterfowl farming industry. Their corresponding pathogens are MDPV and MDGPV, both of which are non-enveloped icosahedral single-stranded DNA viruses. MDPV can cause panting, weak legs, and diarrhea in young Muscovy ducks. GPV infection mainly causes diarrhea, mental fatigue, and anorexia in young Muscovy ducks and goslings. In some cases, autopsy shows fibrinous enteritis, necrosis and shedding of the mucosal membrane in the middle or posterior part of the small intestine, and the formation of sausage-like embolism with coagulated fibrinous exudate. MDPV and MDGPV are mainly transmitted through the respiratory and digestive tracts. For example, they can be transmitted through the excrement of sick waterfowl and then through feed, drinking water and feeding facilities. They can also contaminate breeding eggs and hatcheries, causing mass illness in young Muscovy ducks. The Muscovy duck flock can first be partially infected with MDPV, causing a certain degree of damage to its liver, spleen and other tissues, leading to a decrease in immunity, and then secondarily infected with MDGPV, making the waterfowl parvovirus disease more serious, causing great difficulties for the accurate diagnosis of clinical etiology, and also bringing great difficulties to the disease purification of Muscovy duck farms. Therefore, the establishment of a rapid and accurate detection method for distinguishing the strong and weak toxins of MDPV and MDGPV is of great significance for the diagnosis, prevention and control, and epidemiological investigation of the "three-week disease" and gosling plague of Muscovy ducks.

[0066] Multiplex PCR is an improvement on the traditional PCR principle, that is, multiple pairs of specific primers are added to the same system to amplify multiple target DNA fragments from multiple DNA templates or different regions of the same template. It is of great significance in the clinical differential diagnosis of mixed infectious diseases, especially for the detection of pathogens with similar clinical manifestations and epidemiological characteristics.

[0067] Based on the wide application range, high sensitivity and simple operation of SYBR Green I dye, this study selected the fragments with large differences in the 3'-ITR variable region of the virulent PT strain of MDXPV, the weak D strain of MDXPV and the representative P strain of MDPV as target genes, designed a pair of specific primers, and established a SYBR Green I qPCR method for simultaneous detection of virulent MDXPV, weak MDXPV and MDPV. The difference between virulent MDXPV, weak MDXPV and MDPV in the same sample lies in their unique melting temperatures. The melting temperature of the MDXPV-PT virus strain is about 85.2±0.5℃; the melting temperature of the MDXPV-D virus strain is about 83.8±0.5℃; the melting temperature of the MDPV-P virus strain is about 86.1±0.5℃, while no specific melting peaks are shown for other non-targeted common viruses of Muscovy ducks, and the detection limits are all the lowest detection limits of 1×10 1 The method is simple and easy to use. The results can be obtained in about 1 hour. The stability is good, and the coefficient of variation within and between batches is less than 1%. In addition, 35 clinical samples of diseased Muscovy ducks were tested in this study, and it was found that the consistency between the identification results of the SYBR Green I qPCR method established in this study and the sequencing results was 100%, which proved the accuracy and reliability of the technology. The establishment of this method provides technical support for the purification of Muscovy duck gosling plague and Muscovy duck "three-week disease", and for the early elimination of wild-type positive infected Muscovy ducks and the establishment of negative purified Muscovy ducks.

Claims

1. A SYBR Green I real-time fluorescence quantitative PCR detection primer for simultaneously identifying and detecting MDGPV virulent, MDGPV mildly virulent and MDPV, characterized in that: The sequences of the primers are as follows: WPV-ITR-F: 5'-GCATGCGCCCGATCTGCCATGAA-3', WPV-ITR-R: 5'-GTGCTTCCCGGTCATGTGTTTCC-3'.

2. Use of the SYBR Green I real-time fluorescence quantitative PCR detection primer as described in claim 1 in the preparation of a kit for simultaneously identifying and detecting MDGPV virulent, MDGPV mildly virulent and MDPV.

3. A kit for simultaneously identifying and detecting MDGPV virulent, MDGPV mildly virulent and MDPV, characterized in that: The kit comprises the SYBR Green I real-time fluorescence quantitative PCR detection primer as claimed in claim 1.

4. A SYBR Green I real-time fluorescence quantitative PCR detection method for simultaneously identifying and detecting MDGPV virulent, MDGPV mildly virulent and MDPV using the SYBR Green I real-time fluorescence quantitative PCR detection primers according to claim 1, wherein the method is not for the purpose of diagnosis, and is characterized in that: The PCR reaction system is as follows: 20 μL system contains the following components: 2× TopGreen qPCR SuperMix 10μL, 0.5μL of primers WPV-ITR-F and WPV-ITR-R at a concentration of 10μmol / L, 1μL of positive templates corresponding to the three viruses of MDGPV, MDGPV and MDPV, or 3μL of template DNA of the sample to be tested, and make up to 20μL with Nuclease-free Water; The PCR amplification conditions were: 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, for 40 cycles, while collecting the fluorescence signal and making the corresponding melting curve.

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