水泡性口炎病毒双重荧光RT-PCR鉴别检测方法的建立

Development of a duplex real-time RT-PCR assay for the detection of vesicular stomatitis virus

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。

To establish a rapid,sensitive and specific assay for simultaneous detection and differentiation of vesicular stomatitis virus（VSV）,a duplex real-time fluorescent RT-PCR assay was developed with two pairs and two probes of primers designed according to the L gene of vesicular stomatitis Indiana virus（VSV-IND） and vesicular stomatitis New Jersey virus（VSV-NJ）.Under the optimized reaction conditions,VSV-IND and VSV-NJ were specifically detected simultaneously by the assay without cross-reaction with other inactivated virus antigens, including FMDV,BTV,BVDV,SVDV,CSFV and PRRSV RNA.Compared with the conventional RT-PCR,the assay was fast and sensitive.Moreover this assay had good reproducibility,with the coefficient of variation of Ctvalues less than 3%.A total of 126 clinical samples were tested by the established assay and conventional RT-PCR,and the test results were consistent.In conclusion,the established assay is rapid,specific and sensitive,and is useful for detection of VSV-IND and VSV-NJ simultaneously.