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当前位置: 首页 > 文章 > 猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪A型轮状病毒多重RT-PCR方法的建立及其应用 中国兽医学报 2016,36 (2) 216-220+227
Position: Home > Articles > Establishment and application of a multiplex reverse transcription-PCR for detecting porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus Chinese Journal of Veterinary Science 2016,36 (2) 216-220+227

猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪A型轮状病毒多重RT-PCR方法的建立及其应用

作  者：
李丽敏;朱卫霞;王晓波;魏艳秋;张鹤;张义明;宋勤叶
单  位：
河北农业大学动物医学院
关键词：
猪流行性腹泻病毒;猪传染性胃肠炎病毒;猪A群轮状病毒;多重RT-PCR
摘  要：
为了建立一种能够同时检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪A群轮状病毒(RVA)的敏感、特异的多重RT-PCR(mRT-PCR)方法,本试验分别针对PEDV和TGEV的N基因、RVA的VP7基因设计了4对引物(其中针对PEDV N基因的引物为内、外2对),结合mRT-PCR和PEDV的套式RT-PCR(nRTPCR),建立了检测PEDV、TGEV和RVA的mRT-PCR方法。mRT-PCR能够同时扩增出针对3种病毒的预期大小的目的核苷酸片段,与猪繁殖与呼吸综合征病毒、猪瘟病毒、伪狂犬病毒及猪圆环病毒2型等病毒无交叉反应;可以检测到PEDV、TGEV和RVA的最低核酸浓度分别为16.07、803.9和321.5μg/L。应用mRT-PCR检测了31份腹泻仔猪的血清、肛拭子或小肠组织,其中PEDV、TGEV与RVA的阳性率分别为61.29%、6.45%和25.81%,并检测到了PEDV与RVA或TGEV双重感染及PEDV、TGEV和RVA三重感染的样品。在检测临床样品时,mRT-PCR和单项RT-PCR检出PEDV、TGEV和RVA的符合率分别为96.77%、93.54%和93.54%。上述结果表明,mRTPCR能够快速鉴别检测PEDV、TGEV和RVA 3种病毒,PEDV是引起当前仔猪腹泻的主要病原。
译  名：
Establishment and application of a multiplex reverse transcription-PCR for detecting porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus
作  者：
LI Li-min;ZHU Wei-xia;WANG Xiao-bo;WEI Yan-qiu;ZHANG He;ZHANG Yi-ming;SONG Qin-ye;College of Veterinary Medicine,Agricultural University of Hebei;
关键词：
porcine epidemic diarrhea virus;;porcine transmissible gastroenteritis virus;;porcine group A rotavirus;;multiplex RT-PCR
摘  要：
In order to establish a sensitive and special multiplex reverse transcription-PCR(mRTPCR)method for detecting porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV)and porcine group A rotavirus(RVA)simultaneously,four pairs of primers targeting the N gene of PEDV and TGEV,and VP7 gene of GAR were designed by primer design software(Premier 5.0)respectively,in which primers targeting N gene of PEDV included two pairs of internal and external primers.A multiplex RT-PCR combined with PEDV-nest PCR for detecting PEDV,TGEV and GAR was developed using these primers.The expected target gene segments of PEDV,TGEV and GAR were amplified by mRT-PCR,and non-specific amplifications with porcine reproductive and respiratory syndrome virus,classical swine fever virus,porcine pseudorabies virus and porcine circovirus type 2were not occurred.The RNA concentrations of PEDV,TGEV and GAR detected were 16.07,803.9and 321.5μ/L,respectively.Furthermore,thirty-one samples of serum,feces or small intestines from piglets with diarrhea were detected by mRT-PCR.In these clinical samples,the positive percent of PEDV,RVA or TGEV was61.29%,25.81%and 6.45%respectively,as well as double infection,and triple infection of PEDV,RVA and TGEV were founded also.In addition,the coincidences between mRT-PCR and single RT-PCR were 96.77%in PEDV,93.45%in RVA and 93.45%in TGEV.These results showed that mRT-PCR established in this study could simultaneously and rapidly identify PEDV,TGEV and GAR,and indicated that PEDV is the main pathogen causing diarrhea of piglets currently.

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