毛皮兽阿留申病毒SYBR Green Ⅰ-qPCR检测方法的建立及病毒种鉴别

Development and application of SYBR Green Ⅰ qPCR for the detection of fur animal amdoparvovirus

为建立快速定量检测、鉴别毛皮兽阿留申病毒的SYBR Green ⅠqPCR方法,以阿留申病毒属内病毒序列为参考,在VP2序列高变区的保守区段设计合成1对引物。结果,该方法检测水貂阿留申病毒(AMDV)、狐貉阿留申病毒(RF AV)的最低浓度分别为5.38×10~2copies/μL、5.93×10~2copies/μL,且通过熔解曲线可区分鉴别不同病毒种。对55份临床样品以及4株RFAV进行定量检测,结果,有13份PCR检测为阴性的样品被qPCR检测为阳性,说明qPCR检测方法的灵敏度优于普通PCR。上述结果表明,本试验建立的方法是一种高敏感性、特异性检测毛皮兽阿留申病毒,并可通过实测Tm值区分阿留申病毒的分子诊断技术。

To establish a rapid quantitative detection and identification method,SYBR Green I q PCR for fur animal amdoparvovirus,a pair of primers was designed and synthesized.It was located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.In result,the minimum concentrations of Aleutian mink disease virus(AMDV) and raccoon dog and arctic fox amdoparvovirus(RFAV) were 5.38×10~2 copies/μL and 5.93×10~2 copies/μL respectively,and the virus was clearly distinguished by the melt curve.The q PCR testing of 55 clinical specimens and 4 strains of RFAV have been conducted.It was found that 13 samples were negative in PCR but positive in q PCR,indicating that the sensitivity of q PCR is superior to ordinary PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which can be used to clearly distinguish different amdoparvoviruses by measuring Tm of viral amplicon.