摘要
旨在建立一种同时检测猪流行性腹泻病毒(PEDV)、猪肠道病毒9型(PEV-9)、猪嵴病毒(PKV)的三重RT-PCR法,并初步应用于临床样品检测。根据GenBank中PEDV和PKV基因序列保守区设计2对引物,参照文献合成1对PEV-9引物,在同一体系进行RT-PCR扩增,对引物浓度、退火温度、灵敏度、特异性等进行优化,应用该法对采自四川省6个猪场46份样品进行检测。结果表明引物量分别为PEDV 0.5μL、PEV-9 0.25μL、PKV0.25μL,退火温度为55℃时扩增效果最理想;本方法最低检测量分别为PEDV 5.97pg、PEV-9 0.11pg、PKV0.74pg;用该方法对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒、猪萨佩罗病毒、猪博卡病毒、猪环曲病毒、猪源大肠杆菌、猪源沙门菌、副猪嗜血杆菌、猪链球菌和空肠弯曲杆菌等病原进行RT-PCR扩增时,均无非特异性扩增条带出现。对46份腹泻样品的检测结果显示,PEDV感染率为76.1%、PEV-9为73.9%、PKV为39.1%,3种病原在同一样本检出率达29.1%,表明存在混合感染或协同致病的可能;与单项RT-PCR法相比,阳性样本的检出符合率均在91%以上,表明该方法具较高可信度。建立的方法可快速、准确地检测3种猪腹泻病毒性病原,为病原学诊断和分子流行病学调查提供了有效技术支持。
The aim of this study was to establish a triplex for simultaneously detecting porcine epidemic diarrhea virus(PEDV),porcine enterovirus-9(PEV-9),and porcine kobuvirus(PKV). Two pairs of special primers were designed according to the published sequences of PEDV,PKV in GenBank,one primers of PEV-9were synthesized according to reference.A mixture of 3paris of primers was used for amplification of viral nucleic acids,yielding 3different amplicons with sizes of 681bp,454bp,and 313bp for PEDV,PKV and PEV-9respectively by optimizingthe reaction condition.Testing of the sensitivity of multiplex RT-PCR indicated that the lowest detection limits were 5.97pg for PEDV,0.11pg for PEV-9,0.74pg for PKV,respectively.For the specificity test,all of pathogens in the negative controls were found no amplicons.A total of 46specimens from piglets with acute diarrheas collected from Sichuan province were tested by triplex RTPCR,and the positive rate were as follows:PEDV 76.1%,PKV 39.1%,PEV-9 73.9%,and thepositive accordance rate between simplex and triplex PCR of PEDV,PEV-9,PKV were 100%, 91%,and 94%respectively.In addition,the fact that three pathogens were detected simultaneously in one sample indicated mixed infection in the diarrhea disease.It was also suggested that the pathogenic mechanisms of three kinds of pathogens is somehow synergetic.This study showed that the triplex RT-PCR may be a useful tool for rapid and sensitive etiological diagnosis for acute viral diarrheas in piglets and provides an effective technical support for pathogenic molecular epidemiology investigation.Besides,it established the foundation that we can further explore the relationship between the traditional diarrhea pathogens and potential new intestinal pathogens.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2014年第4期603-608,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
"十二五"国家高技术研究发展(863)计划项目(2012AA101304)
西南民族大学中央高校基本科研业务费专项资金项目(2014NZYQN42)
四川省教育厅创新团队项目(13TD0057)
西南民族大学研究生学位点建设项目(2014XWD-S0906)
