猪流行性腹泻病毒强毒株和疫苗株多重RT-PCR鉴别检测方法的建立及应用

发布时间：2018-05-14 22:40

本文选题：猪流行性腹泻病毒 + 野毒株　；参考：《中国兽医科学》2017年01期

【摘要】：为建立猪流行性腹泻病毒(PEDV)的快速鉴别检测方法,针对PEDV基因序列设计3对特异性引物。第1对引物扩增野毒株(经典株、变异株)和疫苗株ORF3基因198 bp和148 bp片段;第2对引物扩增经典株和疫苗株S基因429 bp片段;第3对引物扩增变异株S基因274 bp片段。经过反应条件的优化,建立了能够同时检测并区分PEDV经典株、变异株及疫苗株的多重RT-PCR检测方法。该方法可以特异地检测PEDV,而与猪的其他重要病原FMDV、CSFV、PRRSV、TGEV、PRo V、PCV2、PRV无交叉反应;对重组质粒标准品的检出下限为2.62×10~2copies/L;重复试验获得了均匀一致的结果。应用该方法检测336份临床病料,PEDV阳性的120份,其中变异株96份、疫苗株21份、变异株和疫苗株混合感染3份。结果表明,本研究所建立的多重RT-PCR检测方法特异性强、敏感性高、重复性好,可用于PEDV经典株、变异株和疫苗株的快速鉴别检测和流行病学调查。
[Abstract]:In order to establish a rapid method for identification and detection of porcine epidemic diarrhea virus (PEV), three pairs of specific primers were designed for PEDV gene sequence. The first pair of primers amplified 198bp and 148bp fragments of ORF3 gene from wild virus strain (classical strain, variant strain) and vaccine strain, the second pair of primers amplified S gene 429bp fragment of classical strain and vaccine strain, and the third pair of primers amplified S274bp fragment of mutant strain. After optimization of reaction conditions, a multiplex RT-PCR detection method was established to detect and distinguish PEDV classical strains, variant strains and vaccine strains simultaneously. This method can specifically detect PEV, but has no cross reaction with other important pathogens of swine, such as FMDV / CSFV, PRRV / TGEV / PRO / VGV _ 2 / PRV, and the detection limit of recombinant plasmid standard is 2.62 脳 10 ~ (-2) copi _ (2) / L / L, and the result of repeated test is uniform and consistent. The method was used to detect 120 cases of PEDV positive in 336 clinical samples, including 96 variant strains, 21 vaccine strains and 3 mixed infection strains. The results showed that the multiplex RT-PCR assay developed in this study was highly specific, sensitive and reproducible, and could be used for rapid identification and epidemiological investigation of classical PEDV strains, variant strains and vaccine strains.
【作者单位】：广西动物疫病预防控制中心;广西大学动物科学技术学院;
【基金】：广西水产畜牧科技项目(桂渔牧科201528017) 广西科学研究与技术开发计划项目(桂科转14125004-22)
【分类号】：S852.651

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