| Porcine circovirus disease,porcine diarrhea virus disease and porcine vesicular disease are important diseases that seriously affect the healthy development of pig industry in China.Because the clinical symptoms of the diseases caused by three groups of pathogens of porcine circovirus,porcine diarrhea virus and porcine vesicular virus are very similar,it is difficult to distinguish them effectively in clinic.Therefore,laboratory detection methods are needed to realize simultaneous differential diagnosis.Aiming at the common pathogens of Porcine circovirus disease,porcine diarrhea and porcine vesicular disease,a multiplex fluorescent PCR identification method for detecting three or four pathogens at the same time was established,and the genetic evolution of some porcine rotavirus epidemic strains in pigs in Hebei province was analyzed.1.In order to realize the simultaneous detection and differentiation of porcine circovirus 2(PCV2),porcine circovirus 3(PCV3)and porcine circovirus 4(PCV4),the specific primers and Taq Man probes were synthesized based on the conserved regions of PCV2 Rep gene,PCV3 Cap gene and PCV4 Cap gene,and a triple real-time PCR for simultaneous,rapid,efficient and differential detection of PCV2,PCV3 and PCV4 was established through optimizing the reaction conditions.The results showed that this method only showed positive amplifications for PCV2,PCV3and PCV4,and had no cross-reactions with other common porcine pathogens used in this study such as PRV,PPV,PRRSV,CSFV,M.hyo,PEDV and TGEV were detected.The limit of detection was 1.69×10~1 copies for PCV2,1.38×10~2 copies for PCV3 and1.42×10~2 copies for PCV4.The assay had good reproducibility with intra-and-inter assay coefficients of variation at less than 1.6%.The developed triple real-time PCR assay for PCV2,PCV3 and PCV4 was further applied to detect 363 swine clinical samples from farms,slaughterhouses and harmless treatment plants in different areas of Hebei Province.The detection results showed that 184 of the positive rates of samples were PCV2 were positive 50.69%(184/363),65 of the samples were PCV3positive 17.91%(65/363),and 21 the of the samples were PCV4 positive 5.78%(21/363).Among the positive samples,3 samples were co-infected with PCV2,PCV3and PCV4(0.83%).In summary,the PCV2,PCV3 and PCV4 triple real-time PCR assay developed in this study could effectively realize the simultaneous and rapid detection of PCV2,PCV3 and PCV4 in clinical samples.2.In order to realize the simultaneous detection and differentiation of porcine delta coronavirus(PDCo V),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(Po RV)and porcine epidemic diarrhea virus(PEDV),the specific primers and Taq Man probes were synthesized based on the conserved regions of PDCoV M gene,TGEV S gene,PoRV VP6 gene and TGEV N gene,and a quadruple real-time RT-PCR assay for simultaneous detection of PDCo V,TGEV,Po RV and PEDV was established through optimizing the reaction conditions.The results showed that the developed quadruple real-time RT-PCR assay only showed positive amplifications for PDCo V,TGEV,Po RV and PEDV,and no cross-reactions with other common porcine pathogens used in this study such as PRV,PPV,PRRSV,CSFV and PCV2 were observed.The limit of detection of the quadruple real-time RT-PCR assay was 1.17×10~3 copies for PDCo V,1.13×10~3 copies for TGEV,1.31×10~2 copies for Po RV and 1.18×10~2 copies for PEDV.The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 4.5%.The developed quadruple real-time RT-PCR assay was further applied to detect the PDCo V,TGEV,Po RV and PEDV in 199 swine clinical samples collected from different regions of Hebei province.The detection results showed the positive rates of Po RV were 11.56%(23/199),the positive rates of PEDV were 6.03%(12/199),and the positive rates of TGEV were 0.50%(21/363)The detection results showed that 23 of the samples were Po RV positive(11.56%),12 of the samples were PEDV positive(6.03%)and 1 of the samples were TGEV positive(0.50%),in which all the samples were single infection and no PDCo V was detected.In conclusion,the quadruple real-time RT-PCR assay developed in this study could realize the simultaneous detection and differentiation of PDCo V,TGEV,Po RV and PEDV,and provided an effective technical support for the rapid detection and differentiation of porcine viral diarrhea in the laboratory.3.In order to realize the simultaneous detection and differentiation of foot and mouth disease virus(FMDV),vesicular stomatitis virus(VSV),swine vesicular disease virus(SVDV)and senecavirus A(SVA),the specific primers and Taq Man probes were synthesized based on the conserved regions of FMDV 3D gene,VSV N gene,SVDV VP1 gene and SVA 3D gene,and a quadruple real-time RT-PCR assay for simultaneous detection of FMDV,VSV,SVDV and SVA was established through optimizing the reaction conditions.The results showed that the developed quadruple real-time RT-PCR assay only showed positive amplifications for FMDV,VSV,SVDV and SVA,and no cross-reactions with other common porcine pathogens used in this study such as PRV,PPV,PRRSV,CSFV and PCV2 were observed.The limit of detection of the quadruple real-time RT-PCR assay was 1.45×10~2copies for FMDV,1.32×10~3copies for VSV,1.30×10~2copies for SVDV and 1.04×10~2 copies for SVA.The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 6.0%.The established quadruple real-time fluorescent RT-PCR assay methods of FMDV,VSV,SVDV and SVA were further applied used to detect220 tissue samples from diseased dead pigs in a pig farm in different regions of Hebei Province.The results were negative.The quadruple real-time fluorescent RT-PCR detection method established in this study realizes the simultaneous and rapid identification and detection of FMDV,VSV,SVDV and SVA.4.Two strains of PORV VP6 gene were obtained after amplification and sequencing of VP6 gene in some positive samples of Po RV.Homology analysis showed that the nucleotide and amino acid sequence homology between the two Po RV VP6 epidemic strains in Hebei province were 89.4%and 96.2%respectively,which were on the same branch as group A porcine rotavirus. |
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