猪病毒性腹泻病相关病原的MLPA检测技术的建立及应用  

Establishment and Application of Multiplex Ligation-dependent Probe Amplification Assay for Identification of Pathogens Causing Porcine Viral Diarrhea Diseases

作　　者：何海健[1] 刘正奎 吴瑗[1] 王志鹏 陈琳 王磊 周莹珊 蒋春燕[1] 宋厚辉 王晓杜 HE Hai-Jian;LIU Zheng-Kui;WU Yuan;Wang Zhi-Peng;CHEN Lin;WANG Lei;ZHOU Ying-Shan;JIANG Chun-Yan;SONG Hou-Hui;WANG Xiao-Du(College of Agricultural and Biological Engineer,Jinhua Polytechnic,Jinhua 321007,China;College of Animal Science and Technology·College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]金华职业技术学院农业与生物工程学院,金华321007 [2]浙江农林大学动物科技学院·动物医学院,杭州311300

出　　处：《农业生物技术学报》2020年第9期1699-1710,共12页Journal of Agricultural Biotechnology

基　　金：金华市农业类重点项目(2018-2-004;2019-2-012);国家自然科学基金面上项目(31972656);浙江省科技重点研发计划项目(2018C02028);浙江省自然科学基金资助项目(LY16C180001)。

摘　　要：每年冬春季爆发于规模化养猪场的腹泻常由不同病毒引起,临床表现为相似腹泻症状。为了区分临床腹泻样本中的病毒性病原体,迫切需求建立一种快速同时检测常见6种病毒性腹泻疾病的病原技术。本研究针对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S基因、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)N基因、猪Delta冠状病毒(Porcine delta coronavirus,PDCoV)N基因、猪博卡病毒(Porcine bocavirus,pBCaV)NS1基因、猪诺如病毒(Porcine norovirus,pNoV)RdRp基因、猪轮状病毒(Porcine rotavirus,pRV)NSP1基因的核酸保守区域,设计多重连接探针扩增技术(multiplex ligationdependent probe amplification,MLPA)探针和预扩增引物,利用MLPA技术和毛细管电泳技术,建立检测6种疾病病原核酸的检测方法。结果表明,单一探针检测混合模板,显示出高度特异性;混合探针总浓度在1.33 nmol/L时,各种疾病扩增条带具有特异性且与预期大小一致,pBCaV、pNoV、PDCoV、PEDV、pRV、TGEV分别约为102、110、117、124、131、138 bp。本方法与其他临床疾病如猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(Classic swine fever virus,CSFV)、伪狂犬病毒(Pseudorabies virus,PRV)和猪圆环病毒2型(Porcine circovirus type 2,PCV2)没有交叉反应,特异性佳。pBCaV、pNoV、PDCoV、PEDV、pRV、TGEV检测最低极限值分别为:7.58×10^1、7.56×10^0、7.54×10^0、7.53×10^0、7.50×10^1和7.49×10^0 copies/µL。组间和组内重复性好,利用本方法检测临床收集和模拟样本67份,PEDV与病毒分离方法100%符合,pRV、TGEV、pBCaV、pNoV、PDCoV与病毒模拟样本符合率达到100%。本研究为临床提供一种新型的可同时检测6种腹泻病原的技术,为临床疾病防控快速反应提供技术支持。Diarrhea of piglets in large-scale farms is often caused by different viruses,which have similar clinical symptoms in winter and spring.In order to distinguish the pathogens in clinical samples of viral diarrhea,it is urgent to establish a rapid and simultaneous detection technology for viral nucleic acid of six viral diarrhea diseases.In this study,the probes and pre-amplification primers for multiple restriction probe amplification(MLPA)were designed countering for the conserved regions of nucleic acids of Porcine epidemic diarrhea virus(PEDV)S gene,Transmissible gastroenteritis virus(TGEV)N gene,Porcine delta coronavirus(PDCoV)N gene,Porcine Bocavirus(pBCaV)NS1 gene,Porcine norovirus(pNov)RdRp gene,Porcine rotavirus(PRV)NSP1 gene.The method of detecting nucleic acid of 6 diseases was established using MLPA and capillary electrophoresis.The results showed that the detection of the 6 mixed templates by a single probe showed a high specificity.When the total concentration of the mixed probes were 1.33 nmo/L,and the specific amplified bands of various diseases were the same as expected,with the products of pBCaV(102 bp),pNov(110 bp),PDCoV(117 bp),PEDV(124 bp),pRV(131 bp)and TGEV(138 bp).This method had no cross reaction with other clinical diseases about Porcine reproductive and respiratory syndrome virus(PRRSV),Classic swine fever virus(CSFV),Pseudorabies virus(PRV)and Porcine circovirus type 2(PCV2).The minimum limit values for detecting nucleic acid of pBCaV,pNoV,PDCoV,PEDV,pRV,TGEV were 7.58×10^1,7.56×10^0,7.54×10^0,7.53×10^0,7.50×10^1 and 7.49×10^0 copies/µL,respectively.The repeatability between and within groups was well.The results of 67 clinical and simulated samples by this method showed that PEDV were 100%in accordance with the virus isolation method,pBCaV,pNoV,PRV,TGEV and PDCoV were 10^0%in accordance with simulated virus.This study provides a new technology for detecting 6 clinical diarrhea pathogens simultaneously,and for the rapid response of clinical disease prevention and control.

关 键 词：猪病毒性腹泻 多重连接探针扩增技术(MLPA) 毛细管电泳 灵敏度 

分 类 号：S855.3[农业科学—临床兽医学]