羊传染性脓疱病毒LAMP检测方法的建立与应用

Establishment of LAMP detection method for orf virus

为建立一种操作便捷、灵敏度高、特异性强且便于基层兽医检测羊传染性脓疱病毒(ORFV)的方法,本研究基于ORFV-VIR基因设计了外引物F3、B3和内引物FIP、BIP共2对引物,对其进行扩增并连接至载体pMD19-T,构建阳性质粒pMD19-T-VIR。通过设置60℃~67℃温度梯度,比较优化了检测ORFV的LAMP反应条件。比较普通PCR、RT-PCR与LAMP这三种检测方法的特异性和敏感性,选择最优检测方法应用于后续临床样品的检验。结果显示,LAMP检测63℃下孵育40 min,羟基萘酚蓝可通过肉眼直接观察测定结果;特异性结果显示,ORFV与山羊痘病毒(GTPV)、绵羊痘病毒(SPPV)、牛O型口蹄疫病毒(FMDV)的核酸均无交叉反应;敏感性分析LAMP最低检测量是PCR的10倍,为5.1×10^(1)copies/μL,与RT-PCR相同。临床样品检测结果显示,LAMP阳性检测率为38.346%,远高于普通PCR的阳性率(13.46%)。结果表明,LAMP法具有检测操作简便、灵敏高、耗时少等优点,适合基层检测,可作为ORFV临床检测的候选方式。

In order to research an easier,rapid,sensitive and specific diagnostic method is urgently required to detect the sheep infectious impetigo virus(ORFV).In this study,we developed a loop-mediated isothermal amplification(LAMP) the assay for the visual detection of ORFV and evaluated its sensitivity,specificity,and applicability in clinical samples.This assay’s results can be directly visualized by the naked eye using hydrox-ynaphthol blue after incubation for 40 min at 63 ℃.The assay specifically amplified VIR of ORFV DNA and no other viral nucleic acids.The sensitivity of the assay was 10 times more sensitive than ordinary PCR(PCR) and comparable to real-time PCR(RT-PCR).Clinical evaluation revealed that the ORFV detection rate in individual sheep samples and at the farm level was 38.346%,which were higher than ordinary PCR(13.46%).In conclusion,cumulatively,owing to the advantages of high sensitivity and specificity,direct visual monitoring of the results,no possibility for cross-contamination,and being low-cost equipment,the developed LAMP assay will be a valuable tool for the detection of the novel ORFV in clinical samples,even in resource-limited laboratories.