7种猪呼吸道综合征疾病病原多重PCR方法的建立  

Development of multiplex PCR assay for the detection of 7 pathogens of swine respiratory diseases

作　　者：任梅渗 冷依伊 蒙正群 张鹏飞[1,2] 杨泽晓[1] 姚学萍[1] 王印[1,2] REN Meishen LENG Yiyi Meng Zhengqun Zhang Pengfei YANG Zexiao YAO Xueping WANG Yin(College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]动物疫病与人类健康四川省重点实验室,成都611130

出　　处：《中国农业大学学报》2017年第7期47-53,共7页Journal of China Agricultural University

基　　金：国家"十二五"国家科技支撑计划(No.2013BAD12B04)

摘　　要：为建立能够同时检测7种导致猪呼吸道综合症疾病病原的多重PCR方法,参考伪狂犬病病毒(PRV)、猪瘟病毒(CSFV)、非洲猪瘟病毒(ASFV)、猪繁殖与呼吸综合征病毒(PRRSV)、副猪嗜血杆菌(HPS)、多杀性巴氏杆菌(PM)以及胸膜肺炎放线杆菌(APP)标准毒株序列,选择各病毒和细菌的保守序列分别设计7对特异性引物进行多重PCR。各项优化试验结果表明,当反应的退火温度为51℃,各条引物浓度均为0.8μmol/L时扩增的目的条带效果最佳;用敏感性试验检测该反应中各病毒的最小检出量分别为:1.01×10~2(PRV)、1.48×10~3(CSFV)、1.07×10~4(ASFV)、9.73×10~3(PRRSV)、1.82×10~3(HPS)、1.65×10~3(PM)和3.64×10~3(APP)copies/μL。在最优化的反应条件下进行特异性试验,结果未出现交叉反应,能检出对应病原的多种血清型,阴性对照均无非特异性扩增。该方法快捷、灵敏、特异性高,可为临床诊断与流行病学研究提供科学依据。A multiplex PCR method was developed for detection of 7 swine respiratory diseases in this study.Based on the conservative sequences of Porcine pseudorabies virus（PRV）,Classical swine fever virus（CSFV）,Africa swine fever virus（ASFV）,Porcine reproductive and respiratory syndrome virus（PRRSV）,Haemophilus parasuis（HPS）,Pasteurella multocida（PM）,and Actinobacillus pleuropneumoniae（APP）,7pairs of specific primers were designed and synthesized.After optimizing the amplification conditions,the results demonstrated that this assay react best at the annealing temperature of 51 ℃,and the primer concentration of 0.8 μmol/L.The sensitivities of multiplex PCR were 2.59×10~3,2.56×10~3,2.52×10~1,2.49×10~4,2.46×10~3,2.43×10~3 and 2.40×10~1 copies/μL for PRV,CSFV,ASFV,PRRSV,HPS,PM and APP,respectively.Under the optimized reaction conditions,no cross reaction was detected between these 7 kinds of templates and negative control.Moreover,this assay was also effective in the detection of different serotypes in relevant pathogens.In conclusion,the multiple PCR assay is rapid,sensitive and specific,which can provide scientific basis for clinical differential diagnosis and epidemiologic survey.

关 键 词：猪 呼吸道疾病 多重PCR 检测方法 

分 类 号：S856.3[农业科学—临床兽医学]