水泡性口炎病毒双重荧光RT-PCR鉴别检测方法的建立

发布时间：2018-12-16 18:15

【摘要】：为建立水泡性口炎病毒印第安纳型(VSV-IND)和新泽西型(VSV-NJ)的快速鉴别检测方法,本研究根据水泡性口炎病毒2种血清型相对保守的L基因为靶序列,分别设计2对型特异性引物和2条不同荧光基团修饰的探针。经过对反应体系的优化,建立了能够同时检测VSV-IND和VSV-NJ的双重荧光RTPCR方法。该方法能准确鉴别VSV两种血清型,与口蹄疫病毒(FMDV)、蓝舌病病毒(BTV)、牛病毒性腹泻病毒(BVDV)、猪水泡病病毒(SVDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)等灭活抗原核酸无非特异性扩增,特异性强。与普通RT-PCR相比,反应快速、灵敏度高。Ct值的变异系数小于3%,重复性好。利用所建立方法对126份临床样品进行检测,结果与普通RT-PCR相同。上述结果表明,本研究建立的方法为VSV-IND和VSV-NJ的同时鉴别检测提供了一种快速、特异和敏感的方法。
[Abstract]:In order to establish a rapid method for the identification and detection of vesicular stomatitis virus (VSV-IND) and New Jersey virus (VSV-NJ), two serotypes of vesicular stomatitis virus (BMDV) were selected as target sequences. Two pairs of type specific primers and two probes modified by different fluorescent groups were designed. After optimizing the reaction system, a double fluorescence RTPCR method was established for simultaneous detection of VSV-IND and VSV-NJ. This method can be used to identify the two serotypes of VSV accurately, which can be compared with (FMDV), blue tongue disease virus, (BTV), bovine viral diarrhea virus, (BVDV), virus, porcine vesicular disease virus, (SVDV), swine fever virus (CSFV),. Inactivated antigen nucleic acids such as porcine reproductive and respiratory syndrome virus (PRRSV) were amplified with high specificity. Compared with ordinary RT-PCR, the reaction was rapid, the sensitivity was high, the coefficient of variation of Ct value was less than 3 and the repeatability was good. The established method was used to detect 126 clinical samples and the result was the same as that of common RT-PCR. The results showed that the method established in this study provides a rapid, specific and sensitive method for simultaneous identification and detection of VSV-IND and VSV-NJ.
【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;
【基金】：国家科技支撑计划项目(2013BAD12B03) “十三五”国家重点研发计划课题项目(2016YFC1200904)
【分类号】：S852.65

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