双重荧光RPA扩增方法快速鉴别两种血清型水泡性口炎病毒

发布时间：2019-01-04 15:40

【摘要】：本研究利用重组酶聚合酶扩增(RPA)技术,根据水泡性口炎病毒印第安纳型(VSV-IND)和新泽西型(VSVNJ)相对保守的L基因为靶序列设计并筛选出两套特异性的引物和探针,建立了快速鉴别检测水泡性口炎病毒两种血清型的双重荧光RT-RPA方法。试验结果表明,该方法特异性强,与猪水泡病病毒(SVDV)、口蹄疫病毒(FMDV)、蓝舌病病毒(BTV)、猪繁殖与呼吸综合征病毒(PRRSV)、牛病毒性腹泻病毒(BVDV)、猪瘟病毒(CSFV)等灭活抗原核酸无交叉反应;灵敏度高,最低可检测核酸浓度为12.7fg/μL;简便快速,反应时间短(15min),且重复性好。利用所建立方法对124份临床样品进行检测,结果与双重荧光RT-PCR一致。本文为VSV-IND和VSV-NJ的快速鉴别检测提供一种新方法,尤其适合现场检疫或基层实验室的快速检测。
[Abstract]:In this study, (RPA) was amplified by recombinant enzyme polymerase. Two sets of specific primers and probes were designed and screened according to the relative conserved L-base of VSV-IND and (VSVNJ) of New Jersey type. A double fluorescence RT-RPA method for rapid identification and detection of two serotypes of vesicular stomatitis virus was established. The results showed that the method was specific to porcine blisters disease virus (SVDV), foot and mouth disease virus (SVDV), (FMDV), bluetongue virus (BTV), porcine reproductive and respiratory syndrome virus (PRRSV), bovine viral diarrhea virus (BVDV),). The inactivated antigen nucleic acid such as (CSFV) of CSFV had no cross reaction. The lowest detectable nucleic acid concentration is 12.7fg/ 渭 L, which is simple, rapid, short reaction time (15min) and reproducible. The established method was used to detect 124 clinical samples and the results were consistent with double fluorescence RT-PCR. This paper provides a new method for rapid identification and detection of VSV-IND and VSV-NJ, which is especially suitable for field quarantine or rapid detection in basic laboratory.
【作者单位】：深圳出入境检验检疫局动植物检验检疫技术中心;中国检验检疫科学研究院;
【基金】：“十三五”国家重点研发计划课题(项目号:2016YFC1200905)题目:重要新发突发病原体应急处置方案与规范研究 “十三五”国家重点研发计划课题(项目号:2016YFD0500708),题目:猪重要疫病分子检测技术研究~~
【分类号】：S852.65

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