猪流行性腹泻病毒变异毒株与经典毒株RT-PCR鉴别检测方法的建立及应用

Development and applications of an RT-PCR method for differentiation between variant porcine epidemic diarrhea virus strains and classical strains

为建立猪流行性腹泻病毒(PEDV)变异毒株的快速RT-PCR检测方法,根据GenBank中PEDV S基因序列设计合成1对特异性扩增引物,通过优化反应条件,建立了检测PEDV变异毒株的RT-PCR方法。结果显示,建立的RT-PCR鉴别检测方法能特异性区分疫苗株CV777和PEDV变异毒株,仅能扩增出PEDV变异毒株826bp的目的片段,与猪传染性胃肠炎病毒、A群猪轮状病毒、猪嵴病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪瘟病毒及猪细小病毒均无交叉反应。敏感性试验结果显示,该方法能检测到的最低核酸质量为11.3pg。利用该方法对采集自广西部分地区的123份临床腹泻样品进行检测,临床腹泻样品中PEDV阳性率为67.5%,变异毒株占总PEDV毒株的86.7%(72/83)。结果表明,该RT-PCR鉴别诊断方法特异性强、灵敏度高、操作简单,为猪流行性腹泻的流行病学及病原诊断研究提供了可供借鉴的技术手段。

In order to establish a rapid RT-PCR assay for detection of variant porcine epidemic diarrhea virus(PEDV)strains,apair of primers was designed based on S gene sequence of PEDV published in GenBank.After optimization of the reaction system,a rapid RT-PCR method was established.In result,a fragment of 826bp was successfully amplified only from variant PEDV by RT-PCR,while the expected target fragment could not be amplified from transmissible gastroenteritis virus,group A porcine rotavirus,porcine kobuvirus,porcine reproductive and respiratory syndrome virus,pseudorabies virus,classical swine fever virus,and porcine parvovirus.Testing of the sensitivity of RT-PCR indicated that as low as 11.3pg nuclear acids could be detected accurately and rapidly.One hundred and twenty-three stool specimens collected from different farms in Guangxi were detected by the established RT-PCR,the positive rate of PEDV was 67.5%,and the positive rate of variant PEDV was 86.7%(72/83).Therefore the RT-PCR could be used as an effective tool for differentiating diagnosis of the highly pathogenic PED in epidemiological investigations.