摘要
为建立快速、特异、灵敏的毛皮兽阿留申病毒可视化交叉引物等温扩增检测方法(CPA),以阿留申病毒属内病毒序列为参考,在VP2序列高变区保守区段设计合成4条特异引物。通过优化CPA反应条件,建立了毛皮兽阿留申病毒可视化交叉引物等温扩增检测方法,并进行了特异性、敏感性试验以及对临床样品的检测。结果显示,该方法可扩增水貂阿留申病毒(AMDV)和狐貉阿留申病毒(RFAV),对犬科和水貂组织以及常见病毒无特异性反应;对AMDV、RFAV可视化检测下限分别为5.38×10~2copies/μL、5. 93×10~2copies/μL;对83份临床样品的检测结果显示,其中有16份PCR检测为阴性,CPA、qPCR检测为阳性的样品,表明CPA检测方法优于传统PCR。本试验建立了一种高敏感性、特异性检测毛皮兽阿留申病毒的方法,通过添加荧光染料实现了可视化检测,它更加适合基层兽医站、养殖场进行快速核酸检测。
To establish a rapid,specific and sensitive method for the detection of fur animal amdoparvovirus visual by using cross-priming amplification(CPA),four primers was designed and synthesized.They were located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.The reaction conditions were optimized,the specificity and sensitivity were determined,and clinical samples were detected.In result,the method may be used to amplify AMDV and RFAV with no cross reaction with canine and mink tissue and common viruses.The minimum concentrations of visual detection for AMDV and RFAV were 5.38×10~2 copies/μ L and 5.93×10~2 copies/μL,respectively.Out of 83 clinical specimens,16 samples are PCR negative,but both q PCR and CPA positive,indicating that the sensitivity of CPA is superior to the traditional PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which is visually detected by fluorochrome.It can provide a widely applicable technique for rapid quarantine,which is more suitable,especially in resource limited laboratories and farms as well as in field conditions.
引文
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