猪伪狂犬病毒纳米PCR检测方法的建立
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S852.651

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科技部国家重点研发计划(2016YFD0501501)


A NanoPCR Assay for Detection of Pseudorabies Virus
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    摘要:

    [目的]快速灵敏检测猪伪狂犬病毒(PRV)[方法]根据PRV gB基因设计特异性引物,建立了PRV纳米PCR检测方法。[结果]PRV纳米PCR方法的最低检出限为10 拷贝/μL,其敏感性比未添加纳米金的对照PCR提高100倍;建立的纳米PCR与猪细小病毒、猪圆环病毒Ⅱ型、猪繁殖与呼吸综合征病毒等常见猪病毒性病原均无交叉反应。应用纳米PCR和PRV国家标准中的PCR方法,对2014—2016年期间采集自17个省市发病猪场的148份临床样品进行平行检测,PRV纳米PCR的阳性检出率为49.3%(73/148),PRV国标PCR方法的阳性检出率为23.6%(35/148),并且PRV纳米PCR检测为阳性,而PRV国标PCR方法检测为阴性的38份样品,测序结果均确认为PRV阳性。[结论]本研究建立的纳米PCR检测方法敏感特异,可用于猪伪狂犬病的病原学检测。

    Abstract:

    [Objective]In order to develop a rapid and sensitive method for detecting Pseudorabies virus (PRV).[Methods]A nanoPCR assay for detection of PRV was developed with a pair of specific primers that were designed based on sequence of gB gene.[Results]Results showed the detection limit of this assay was 10 copies/μL,sensitivity of which was 100 times higher than conventional PCR assay that no gold nanoparticle was added. Good specificity was also identified that there was no cross reaction with other common viral pathogens of swine such as porcine parvovirus(PPV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus (PRRSV). Using PRV nanoPCR and national standard PCR test of gB gene,148 clinical samples collected from 17 different areas during 2014-2016 were conducted parallel detection. The positive rates by nanoPCR and national standard PRV were 43.5%(73/148)and 27.2%(35/148). 38 samples tested negative by national standard PRV presented a positive result when using the method of nanoPCR,which were consistent with DNA sequencing.[Conclusion]The established PRV nanoPCR method was applicable for pathogenic detection of PRV due to its high sensitivity and good specificity.

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张悦勇,南文龙,秦立得,巩明霞,吴发兴,单虎,陈义平.猪伪狂犬病毒纳米PCR检测方法的建立[J].《中国动物检疫》编辑部,2017,34(3):102-105.

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  • 在线发布日期: 2017-03-07
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