猪繁殖与呼吸综合征病毒和盖他病毒双重RT-PCR检测方法的建立与应用  

Development and Application of a Duplex RT-PCR Assay for Detection of PRRSV and GETV

作　　者：王新港[1] 周峰[1] 王傲杰 崔丹丹[1] 常洪涛[1] 陈陆[1] 杨霞[1] 王新卫[1] 王川庆[1] WANG Xingang;ZHOU Feng;WANG Aojie;CUI Dandan;CHANG Hongtao;CHEN Lu;YANG Xia;WANG Xinwei;WANG Chuanqing;(College of Animal Husbandry and Veterinary Science,Henan Agricultural University)

机构地区：[1]河南农业大学牧医工程学院

出　　处：《病毒学报》2018年第2期232-238,共7页Chinese Journal of Virology

基　　金：河南省高校科技创新团队与支持计划资助项目(项目号:14IRTSHN015);题目:动物疫病防控新技术~~

摘　　要：盖他病毒（Getah virus,GETV）可感染猪并引起和猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome,PRRS）相类似的临床表现,且两者可混合感染而难以区分。我国检测PRRSV和其他诸多病毒的多重PCR文献已比比皆是,但尚无同时检测PRRSV和GETV方法的报道。为此,作者根据GenBank中公布的PRRSV和GETV全基因序列,设计了两对分别扩增PRRSV ORF7基因和GETV Cap基因片段的引物,通过反应条件的优化及特异性、灵敏性和重复性试验,首次建立了可同时检测PRRSV和GETV的双重RT-PCR方法。结果显示,该方法可以同时扩增出PRRSV 633bp和GETV 316bp的特异性片段,而对猪瘟病毒等其他相关病毒扩增结果均为阴性。对GETV和PRRSV的cDNA最低检测量分别为2.48×10-4 ng和2.50×10-5 ng。用建立的双重RTPCR方法对河南15个地市92个猪场的136份临床疑似PRRS发病猪样品进行检测,共从58个猪场检出PRRSV单一阳性样品44份,阳性率为32.4%;GETV单一阳性样品7份,阳性率为5.15%;GETV和PRRSV混合感染样品8份,阳性率为5.88%。检测结果与单一RT-PCR结果吻合率达100%。该方法为GETV和PRRSV的快速检测、鉴别诊断和分子流行病学研究等提供了新的技术手段。Getah virus（GETV）is an infectious pathogen affecting humans and animals.The GETV mainly causes the disease in pigs with symptoms similar to those caused by the porcine reproductive and respiratory syndrome virus（PRRSV）.It is difficult to distinguish co-infection of pigs with GETV and PRRSV by clinical means.Several studies based on multiplex polymerase chain reaction（PCR）for detection of PRRSV and other viruses have been reported,but a simultaneous detection methods for PRRSV and GETV have not.Therefore,based on the whole genome sequences of PRRSV and GETV published in GenBank,two pairs of primers for amplification of PRRSV ORF7 and GETV Cap were designed,and a duplex reverse transcription PCR（RT-PCR）assay was established in China for detection of GETV and PRRSV in a single amplification reaction.This method was used to amplify a 633 bp specific fragment of PRRSV ORF7 and 316 bp specific fragment of GETV Cap gene.The nucleic acid amplification,however,was negative for all the genes for the swine fever virus,porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus,porcine parvovirus,porcine pseudorabies virus and porcine circovirus.The limit of detection（in ng）of GETV and PRRSV was 2.48 ×10-4 and 2.50×10-5,respectively.A total of 136 clinical samples collected from 92 farms in 15 cities in Henan Province were tested by duplex RT-PCR.The number of positive samples from 58 farms with PRRSV infection only and with GETV infection only was44（32.4%）and 7（5.15%）,respectively.The number of samples co-infected with both pathogens was 8（5.88%）.Detection by single RT-PCR and duplex RT-PCR was 100% consistent with each other in the triple detection test.This new method could be used for the rapid detection,differential diagnosis and molecular epidemiology research of the diseases caused by PRRSV and GETV.

关 键 词：猪繁殖与呼吸综合症(PRRS) 盖他病毒(GETV) 双重RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]