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

Abstract

The application relates to SYBR Green I real-time fluorescent quantitative PCR detection primers for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV, the sequences of the primers are shown as SEQ ID NO. 1-2, and the primers are applied to the preparation of a MDGPV virulent, MDGPV attenuated and MDPV simultaneous identification and detection kit. The detection method established by using the primers can simultaneously detect MDGPV virulent, MDGPV attenuated and MDPV, has good specificity, does not have cross reactions with other main waterfowl viruses, has strong specificity, and the detection lower limit of pMD-MDGPV PT-ITR, pMD-MDGPV D-ITR and pMD-MDPV-ITR plasmid standard products is 1.0x10 1 copies per mu L; the variation coefficients of the repeatability test of the method in the group and between groups are all less than 1.0%, and the repeatability is good.

Description

TECHNICAL FIELD

[0001] The application relates to a SYBR Green I fluorescent quantitative PCR detection primer for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV and application thereof. BACKGROUND

[0002] Muscovy duck parvovirus disease (commonly known as Muscovy duck "three-week disease") has a morbidity of 27% to 60% and a mortality of 22% to 60%, and most of the recovered ducks become mummies. The pathogen of the disease is Muscovy duck parvovirus (MDPV). In the Muscovy duck breeding area, it is found that in the Muscovy duckling group, an epidemic disease with a morbidity of 50% to 70% and a mortality of 40% to 65% occurs, which is mainly characterized by diarrhea of different degrees and intestinal mucosal shedding to form embolism in part of the sick ducks. The pathogen is a new type of chimeric strain, namely Muscovy duck origin-goose parvovirus (MDGPV), which is generated by natural recombination of classical goose parvovirus (C-GPV) and MDPV. The two infectious diseases are important diseases that seriously affect the Muscovy duck breeding industry in China at present, and they are often mixedly infected clinically, causing great economic losses to the Muscovy duck breeding industry. MDGPV belongs to the Anseriform dependoparvovirus 1 species of the Dependovirus genus of the Parvoviridae family, and there is a certain antigenic cross between the MDGPV of the same species and the MDPV, and the two viruses can also naturally infect Muscovy ducks, so it is not easy to distinguish MDGPV from MDPV in the immunological detection of polyclonal antibodies. In recent years, MDGPV has been continuously mutating, and the recombination and variation among different virus strains have made Muscovy duck origin-goose parvovirus tend to be complicated in clinical manifestations, and often different from the typical pathological changes of Muscovy duck origin-goose parvovirus in pathological examination, causing great difficulty in veterinary clinical diagnosis. At present, the MDGPV attenuated vaccine used in the Muscovy duck breeding area in China has the characteristics of good safety, strong immunogenicity, long duration of immune protection and high immune protection efficiency, and plays a very important role in the prevention and control of Muscovy duck origin-goose parvovirus. However, the application of the MDGPV attenuated vaccine makes the differential diagnosis of MDGPV virulent and attenuated cases complicated in the clinic. In order to cooperate with the purification of MDGPV and MDPV in the Muscovy duck breeding farm, it is particularly important to establish a sensitive, specific and reproducible multiplex PCR detection method for rapidly distinguishing virulent infection from attenuated vaccination.

[0003] Therefore, in order to establish a rapid identification and detection method of MDGPV virulent and attenuated viruses and MDPV, a pair of specific primers is designed according to the difference sequence of 3'-ITR genes of MDGPV parent virulent strain and vaccine strain and MDPV representative strain, and a SYBR Green I qPCR differential detection method for rapidly distinguishing MDGPV virulent, MDGPV attenuated and MDPV is successfully established by using only a pair of specific qPCR primers, which provides an effective technical support for real-time monitoring of MDPV and MDGPV in Muscovy duck population and immune prevention and control. SUMMARY

[0004] The purpose of the present application is to provide a SYBR Green I qPCR detection primer for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV and application, and a SYBR Green I qPCR detection method for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV established by using the primer.

[0005] The purpose of the present application is achieved by the following technical solutions.

[0006] A SYBR Green I qPCR detection primer for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated 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'-GTGCTTCCGGTCATGTGTTTCC-3'(SEQ ID NO.2).

[0008] The application of the SYBR Green I qPCR detection primer in the preparation of a kit for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV.

[0009] The present application also provides a kit for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV, which comprises the SYBR Green I qPCR detection primer.

[0010] The application also provides a SYBR Green I fluorescent quantitative PCR detection method for simultaneously identifying MDGPV virulent virus, MDGPV attenuated virus and MDPV by using the SYBR Green I qPCR detection primer, and the method is not for the purpose of diagnosis, and a PCR reaction system thereof comprises the following components in a 20-mL system: Top Green qPCR SuperMix 10 muL, 0.5 muL of 10-mu mol / L primer WPV-ITR-F and primer WPV-ITR-R respectively, 1 muL of positive templates of the three viruses of MDGPV virulent virus, MDGPV attenuated virus and MDPV or 3 muL of template DNA of a sample to be detected, and Nuclease-free Water is added to 20 muL.

[0011] PCR amplification conditions are as follows: 94 DEG C for 30 s; 94 DEG C for 5 s, 60 DEG C for 30 s, 40 cycles, and fluorescence signals are collected at the same time to make a corresponding melting curve.

[0012] Compared with the prior art, the application has the following advantages:

[0013] The application designs a pair of specific fluorescent quantitative primers according to the difference of highly conserved 3'-ITR gene nucleotide sequences of MDGPV virulent strain, MDGPV attenuated strain and MDPV representative strain, optimizes reaction conditions, takes 3'-ITR genes of recombinant plasmids pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR of MDGPV virulent PT strain, MDGPV attenuated D strain and MDPV-P strain as plasmid standard products, and establishes a SYBR Green I qPCR method for identifying MDGPV virulent virus, MDGPV attenuated virus and MDPV. The results show that the method can simultaneously detect MDGPV virulent virus, MDGPV attenuated virus and MDPV, and has good specificity, and has no cross reaction with other main waterfowl viruses, and has strong specificity; the detection lower limit of the plasmid standard products pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR is 1.0 x 10 1 copies / muL; the variation coefficients of the repeatability test of the method in the group and between groups are less than 1.0%, and the repeatability is good.

[0014] The method was used to detect 35 clinical tissue samples of sick Muscovy ducks, and the positive rates of MDGPV virulent and MDPV were 11.43% and 8.75% respectively, the mixed rate was 8.75%, and MDGPV attenuated was not detected. The detection results of the SYBR Green I fluorescent quantitative PCR method for clinical samples were consistent with the sequencing analysis results.

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

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

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

[0018] Figure 3 Specificity test results of SYBR Green I qPCR, wherein 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 Sensitivity detection of MDGPV-PT, wherein 1-9: 1×10 8 -1×10 0 copies / μL MDGPV-PT positive plasmid, 10: negative control.

[0020] Figure 5 Sensitivity detection results of MDGPV-D, wherein 1-9: 1×10 8 -1×10 0 copies / μL MDGPV-D positive plasmid, 10: negative control.

[0021] Figure 6 Sensitivity detection results of MDPV-P, wherein 1-9: 1×10 8 -1×100 Copy / μL MD PV-P positive plasmid, 10: negative control. DETAILED DESCRIPTION

[0022] The present application will be described in detail below with reference to the accompanying drawings and examples:

[0023] 1. Materials and methods

[0024] 1.1 Main experimental materials

[0025] Muscovy duck reovirus (MDRV), new duck reovirus (NDRV), duck hepatitis virus type I (DHV-I), duck paramyxovirus (DPMV), duck plague virus (DEV), duck tembusu virus (DTMUV), duck short beak and dwarf syndrome virus (SBDS-GPV), classic goose parvovirus (C-GPV), muscovy duck parvovirus (MDPV) standard virus P strain, muscovy duck small goose plague virus (MDGPV) virulent PT strain, muscovy duck small goose plague (MDGPV) attenuated D strain were preserved by the Animal Virus Room of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0026] pMD-18T plasmid, E. coli competent DH5α, etc. were purchased from Baosheng Bioengineering (Dalian) Co., Ltd. Simple Viral DNA / RNA Kit (ER211-01), Top Green qPCR SuperMix (AQ131-01) was purchased from Beijing Zixiaojin Biotechnology Co., Ltd. 2x Rapid Taq Master Mix (P222-01), FastPure Plasmid Mini Kit (DC201-01), FastPure Gel DNA Extraction Mini Kit (DC301-01) were purchased from Novozyme Biotech Co., Ltd. FastKing One-Step Genomic cDNA First-Strand Synthesis Premix (KR118) was purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd. 96 Fluorescence quantitative PCR instrument was purchased from Roche Company, USA.

[0027] 1.2 Primer design

[0028] The 3'-ITR genes of the MDGPV virulent PT strain, the MDGPV attenuated D strain and the MDPV standard virulent P strain are selected as target genes. Through BLAST comparison, nucleotide regions representing the ITR gene sequence characteristics of the MDGPV virulent and attenuated strains and the MDPV are selected. A plurality of primers are designed by using Oligo 6.0 software. Finally, a pair of SYBR Green I qPCR primers (WPV-ITR-F / WPV-ITR-R) are successfully screened through optimization and screening, and the pair of primers are used for constructing a plasmid standard and establishing a detection method. The primers are synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and the specific information is shown in Table 1.

[0029] Table 1 primer information for virus nucleic acid detection

[0030]

[0031] 1.3 Extraction of virus genes

[0032] The MDRV, NDRV, DHV-I, DPMV and DTMUV are extracted by using a virus DNA / RNA genome extraction kit and reverse transcribed into cDNA, and the DEV, SBDS-GPV and C-GPV genome DNA are extracted. The samples are stored at -80℃ for standby use.

[0033] 1.4 Preparation of plasmid standard

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

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

[0036] MDGPV-PT, MDGPV-D and MDPV-P strain positive standard as template, using 20 μL system: Top Green qPCR SuperMix 10 μL, WPV-ITR upstream and downstream primers (10 μmol / L) 1 μL each, 1 μL of positive template, and Nuclease-free Water to 20 μL. The SYBR Green I fluorescent quantitative PCR of MDPV-P and MDGPV-PT, MDGPV-D strains was set at 55℃, 56℃, 57.2℃, 58.5℃, 59.2℃, 60℃, etc. 9 annealing temperature optimization, according to the best annealing temperature after optimization, the concentration of 10 μmol / L of primers in the range of 0.5-2.5 μL volume and the template in the range of 0.5-2.5 μL volume were optimized by matrix method.

[0037] 1.6 Standard curve and melting curve drawing

[0038] MDGPV-PT, MDGPV-D and MDPV-P strain plasmid standard were diluted by 10 times, 1×10 6 copies / μL to 1×10 0 copies / μL of plasmid as template, and each dilution set 3 repeats, and set up negative control group, with the optimized SYBR Green I qPCR method in step 1.5 to amplify. The initial template copy number was used as the abscissa, and the cycle Ct value was used as the ordinate to draw the quantitative standard curve, and the melting curve was generated.

[0039] 1.7 Specificity test

[0040] The DNA of MDPV-P strain, MDGPV-PT strain, MDGPV-D, C-GPV NP5 strain, SBDS-GPV M15 strain, DEV-fj1 strain, and the cDNA of MDRV MW9710 strain, NDRV NP03 strain, DHV-INA strain, DPMV LG strain were used as templates, 3 kinds of recombinant plasmid standard mixture as positive control, Nuclease-free Water as negative control, each sample repeated 3 times, using the established SYBR Green I qPCR method to amplify, to evaluate the specificity of the method.

[0041] 1.8 Sensitivity test

[0042] MDGPV-PT, MDGPV-D and MDPV-P strain plasmid standard were diluted by 10 times, 1×10 8 copies / μL to 1×10 0The plasmid of 9 dilutions per μL was used as template (3 replicates) to verify the sensitivity of the method under the optimized SYBR Green I qPCR reaction condition.

[0043] 1.9 Reproducibility test

[0044] 1 x 10 5 , 1 x 10 4 , 1 x 10 3 copies / μL of 3 dilutions were used as template, 3 replicates were set for each concentration, and the SYBR Green I qPCR method optimized in this test was used to test the 3 reaction systems of the same batch and 3 reaction systems of different batches, and the coefficients of variation of the batch and the batch were calculated respectively to evaluate the reproducibility of the method. A negative control was also set up.

[0045] 1.10 Detection of clinical samples

[0046] The liver, spleen, kidney and small intestine tissue samples of 35 clinically ill Muscovy ducks were taken, cut into small pieces weighing about 15 g, put into 2 mL sterile centrifuge tubes, added with appropriate PBS, repeated freeze-thawing for 3 times, ground by a tissue grinder and centrifuged to take the supernatant to extract total DNA, and the optimized SYBR Green I qPCR was used for detection. The positive samples of amplification were sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequence determination, and the coincidence rate of the PCR method and the sequencing result was calculated to verify the accuracy of the method.

[0047] 2 Results

[0048] 2.1 Identification results of recombinant plasmid standard

[0049] The total DNA of MDGPV-PT, MDGPV-D and MDPV-P strains were extracted as templates, and the target fragments of 3'-ITR genes of MDGPV-PT, MDGPV-D and MDPV-P strains were obtained by PCR amplification with corresponding primers, which were all 100 bp, 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, which were identified by PCR and then sequenced. The sequencing results were compared with the gene sequences of MDGPV-PT, MDGPV-D and MDPV-P viruses published in NCBI, and the homology was 100%, indicating that the positive standard plasmids were successfully constructed. The concentrations of the three recombinant plasmids were determined by ultraviolet spectrophotometer, and converted into copy number (copies / μL) according to the formula. The results showed that 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, respectively. They were all diluted to 1.0×10 9 copies / μL as the recombinant plasmid standard, and stored at -20℃.

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

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

[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 fold dilutions of pMD-MDGPVPT-ITR, pMD-MDGPVD-ITR and pMD-MDPV-ITR, i.e. 1×10 6 copies / μL, 1×10 0 copies / μL and 1×106 copies / μL ~ 1 x 10 0 copies / μL pMD-MDGPV-D-ITR and 1 x 10 6 copies / μL ~ 1 x 10 0 copies / μL pMD-MDPV-ITR as template, standard curve and melting curve of SYBR Green I qPCR were obtained. Linear relationship expression between logarithm of copy number of MDGPV-PT, MDGPV-D, MDPV-P (X) and cycle threshold (Ct) were Y = -3.2455X + 38.982, 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. Correlation coefficient (R 2 ) of three standard curves were all above 0.998. It showed that three plasmid standard samples had good linear relationship between 1 x 10 6 copies / μL ~ 1 x 10 0 copies / μL and their respective Ct values. Figure 1 ) Melting temperature of MDGPV-PT, MDGPV-D, MDPV-P were about 85.2 ± 0.5℃, 83.8 ± 0.5℃, 86.1 ± 0.5℃ respectively. No melting point peak appeared in negative control, while amplification of standard samples were single wave peak, which indicated that there was no pollution in this experiment, and the data obtained had high reliability( Figure 2 ).

[0054] 2.4 Specificity test results

[0055] DNA of MDGPV-PT strain, MDGPV-D strain, MDPV-P strain, DD-GPV NP5 strain, SBDS-GPV M15 strain, DEV-fj1 strain, and cDNA of MDRV MW9710 strain, NDRV NP03 strain, DHV-INA strain, DPMV LG strain were used as templates for amplification by established SYBR Green I qPCR. The results showed that DNA of recombinant plasmid standard samples pMD-MDGPV-PT-ITR, pMD-MDGPV-D-ITR and pMD-MDPV-ITR and MDGPV-PT, MDGPV-D, MDPV-P isolated virus as positive control appeared amplification curve, while DNA or cDNA of other viruses and negative control had no amplification curve( Figure 3 ). It showed that the established SYBR Green I qPCR method had strong specificity.

[0056] 2.5 Sensitivity test results

[0057] The optimized SYBR Green I qPCR method was used to detect serially diluted pMD-MDGPV-PT-ITR, pMD-MDGPV-D-ITR and pMD-MDPV-P-ITR positive plasmid standard templates (1 x 10 8 copies / μL), and the results showed that good "S" shaped amplification curves were obtained, and the lowest detection limit was 1 x 10 1 copies / μL, which proved that the method had good sensitivity. 1

[0058] 2.6 Reproducibility test results

[0059] The established method was used to perform intra-assay and inter-assay reproducibility tests on three serially diluted MDGPV-PT, MDGPV-D and MDPV-P recombinant plasmid standards pMD-MDGPV-PT-ITR, pMD-MDGPV-D-ITR and pMD-MDPV-P-ITR (1 x 10 5 , 1 x 10 4 and 1 x 10 3 copies / μL), and the results showed that the intra-assay and inter-assay coefficients of variation of MDGPV-PT were 0.14% to 0.26% and 0.40% to 0.80%, respectively; the intra-assay and inter-assay coefficients of variation of MDGPV-D were 0.12% to 0.28% and 0.41% to 0.67%, respectively; and the intra-assay and inter-assay coefficients of variation of MDPV-P were 0.14% to 0.28% and 0.45% to 0.60%, respectively (Table 1). The intra-assay and inter-assay coefficients of variation of the three plasmids were all less than 1%, which indicated that the established SYBR Green I qPCR method had good stability.

[0060] Table 2 SYBR Green I fluorescent quantitative PCR detection reproducibility results

[0061]

[0062] 2.7 Clinical sample detection test

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

[0064] 3Discussion

[0065] Muscovy duck "three-week disease" and muscovy duck gosling plague are both highly contagious and high mortality viral infectious diseases that seriously threaten the healthy development of waterfowl breeding industry. Their corresponding pathogens are MDPV and MDGPV, both of which are non-enveloped icosahedral single-stranded DNA viruses. MDPV can cause panting, foot weakness, diarrhea, etc. in muscovy ducklings. GPV infection mainly leads to diarrhea, listlessness, anorexia, etc. in muscovy ducklings and young geese, and part of the cases can be seen fibrous enteritis, mucosal necrosis and shedding in the middle or posterior segment of small intestine, and the formation of fibrous exudate into sausage-like embolism. MDPV and MDGPV are mainly transmitted through the respiratory tract and digestive tract, such as through the excrement of sick waterfowl, and then through feed, water and breeding facilities. They can also contaminate hatching eggs and incubation rooms, causing batch disease in muscovy ducklings. Muscovy duck flock can be first infected with MDPV, causing certain damage to tissues such as liver and spleen, leading to decreased immunity, and then secondary infection with MDGPV, making the epidemic of waterfowl parvovirus disease more serious, causing great difficulty in accurate diagnosis of clinical etiology, and also bringing great difficulty to the realization of disease purification for muscovy duck breeding farms. Therefore, it is of great significance to establish a rapid and accurate detection method for MDPV and MDGPV virulent and attenuated for the diagnosis, prevention and control, and epidemiological investigation of muscovy duck "three-week disease" and muscovy duck gosling plague.

[0066] Multiple PCR is an improvement based on the principle of traditional PCR, that is, adding multiple specific primers in the same system to amplify multiple target DNA fragments of multiple DNA templates or different regions of the same template, which has great significance in the differential diagnosis of mixed infection in clinic, especially for pathogens with similar clinical manifestations and epidemiological characteristics.

[0067] Based on the characteristics of SYBR Green I dye, such as wide application range, high sensitivity and simple operation, a pair of specific primers were designed to establish a SYBR Green I qPCR method for detecting MDGPV virulent, MDGPV attenuated and MDPV at the same time. The difference between MDGPV virulent, MDGPV attenuated and MDPV in the same sample was their unique melting temperature. The melting temperature of MDGPV-PT strain was about 85.2±0.5℃; the melting temperature of MDGPV-D strain was about 83.8±0.5℃; the melting temperature of MDPV-P strain was about 86.1±0.5℃. However, other non-targeted common viruses of Muscovy duck showed no specific melting peak, and the detection limit was 1×10 1 The detection limit of the method was 1×10 1 The detection limit of the method was 1×10 1 In addition, 35 clinical samples of sick Muscovy ducks were detected, and the consistency of the identification results of the SYBR Green I qPCR method established in this study and sequencing results was 100%, which proved the accuracy and reliability of the technology. The establishment of the method provides technical support for the purification of Muscovy duck gosling plague and Muscovy duck "three-week disease", and the early elimination of wild virus positive infected Muscovy duck flock to establish negative purified Muscovy duck flock.

Claims

1. A SYBR Green I real-time quantitative PCR primer for simultaneously identifying and detecting virulent MGPPV, attenuated MGPPV, and MDPV, characterized in that: The sequence of the primer is as follows: WPV-ITR-F: 5'-GCATGCGCCCGATCTGCCATGAA-3', WPV-ITR-R: 5'-GTGCTTCCGGTCATGTGTTTCC-3'.

2. The SYBR Green I real-time fluorescent quantitative PCR detection primer in the preparation of a kit for simultaneously identifying and detecting MDGPV virulent, MDGPV attenuated and MDPV according to claim 1.

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

4. A SYBR Green I fluorescent quantitative PCR detection method for simultaneously identifying MDGPV virulent, MDGPV attenuated and MDPV by using the SYBR Green I real-time fluorescent quantitative PCR detection primer of claim 1, which is not for diagnostic purposes, characterized in that: The PCR reaction system is as follows: in a 20 μL system, the following components are contained: 2× TopGreen qPCR SuperMix 10 μL, 0.5 μL of primer WPV-ITR-F and primer WPV-ITR-R with a concentration of 10 μmol / L, 1 μL of positive templates of MDGPV virulent, MDGPV attenuated and MDPV respectively or 3 μL of template DNA of a sample to be detected, and Nuclease-free Water is added to 20 μL. The PCR amplification conditions are: 94℃ for 30s; 94℃ for 5s, 60℃ for 30s, 40 cycles, collecting the fluorescent signal at the same time, and making the corresponding melting curve.

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