猪流行性腹泻病毒野毒株及弱毒疫苗株RT-PCR鉴别检测方法的建立及应用

发布时间：2018-05-29 05:54

本文选题：猪流行性腹泻病毒 + 野毒株　；参考：《畜牧与兽医》2017年02期

【摘要】：为建立猪流行性腹泻病毒(PEDV)野毒株及弱毒疫苗株快速鉴别检测方法,根据Gen Bank中公布的PEDV野毒株及弱毒疫苗株的ORF3基因序列,在缺失区的两端设计合成1对特异性扩增引物,用以特异性的扩增PEDV ORF3基因片段,根据目的片段大小判断PEDV的毒株类型;通过退火温度等反应条件优化,建立了区分PEDV野毒株和弱毒疫苗株的RT-PCR鉴别检测方法。结果显示:所建立的RT-PCR鉴别检测方法能特异性区分PEDV野毒株和弱毒疫苗株;PEDV野毒株扩增出234 bp目的片段,PEDV弱毒疫苗株扩增出185 bp目的片段;与猪传染性胃肠炎病毒(TGEV)、A群猪轮状病毒(PRo VA)、猪嵴病毒(PKV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪乙型脑炎病毒(JEV)及猪细小病毒(PPV)均无交叉反应;敏感性试验显示,该方法能检测到病毒滴度为1.3×10~3TCID_(50)/mL。利用该方法对采集自广西部分地区93份临床腹泻样品进行检测,临床腹泻样品中PEDV野毒株阳性率为61.29%(57/93),弱毒株阳性率为5.38%(5/93)。结果表明:该RT-PCR鉴别检测方法特异性强、灵敏度高、操作简便,能快速、准确地区分PEDV自然感染野毒株和弱毒疫苗毒株,为猪流行性腹泻的快速诊断及病原分子鉴别检测研究提供了可供借鉴的技术手段。
[Abstract]:In order to establish a rapid method for identification and detection of wild and attenuated strains of porcine epidemic diarrhea virus (PEV), a pair of specific amplified primers were designed and synthesized at both ends of the deletion region according to the ORF3 gene sequence of PEDV wild strain and attenuated vaccine strain published in Gen Bank. The PEDV ORF3 gene fragment was amplified specifically, and the type of PEDV strain was determined according to the size of the target fragment. By optimizing the reaction conditions such as annealing temperature, a method of RT-PCR identification and detection was established to distinguish PEDV wild strain from attenuated vaccine strain. The results showed that the established RT-PCR identification and detection method could specifically distinguish the PEDV wild strain from the attenuated vaccine strain and amplify the 234bp target fragment of PEDV attenuated vaccine strain and amplify the 185bp target fragment of PEDV attenuated vaccine strain. No cross reaction was found with porcine rotavirus (PRO VAV), porcine cristae virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSVV), pseudorabies virus (PRV), swine fever virus (CSFV), porcine circovirus 2 (PCV2), porcine encephalitis virus (JEV) and porcine parvovirus (PPVs). The sensitivity test showed that the virus titer was 1.3 脳 10 ~ (-3) TCID / mL. This method was used to detect 93 clinical diarrhea samples collected from some parts of Guangxi. The positive rate of PEDV field strain was 61.29 / 93% and that of attenuated strain was 5.38% / 93%. The results showed that the RT-PCR method was specific, sensitive, easy to operate, and could be used to distinguish PEDV naturally infected wild strains from attenuated vaccine strains accurately. It provides a useful technique for the rapid diagnosis and molecular identification of porcine epidemic diarrhea.
【作者单位】：广西兽医研究所/广西畜禽疫苗新技术重点实验室;
【基金】：广西水产畜牧科技推广应用项目(桂渔牧科201633041,桂渔牧科201633044,桂渔牧科201633034) 广西南宁市西乡塘区科技项目(2014304)
【分类号】：S852.65

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