牛结节性皮肤病病毒LAMP快检方法的建立与应用

Establishment and application of a LAMP-based rapid diagnostic method for lumpy skin disease virus

本研究旨在建立一种特异性检测牛结节性皮肤病病毒(LSDV)的LAMP快检方法,以用于该病临床早期诊断与监测。笔者根据LSDV的特异性基因序列,设计了数十套LAMP引物,经过筛选最终确定1套反应效率最高的引物,其位于LSDV095基因序列内。以实验室保存的PUC57-LSDV质粒作为阳性质控进行方法的优化与验证,基于不同的检测需求,分别建立了实时浊度LAMP检测方法、实时荧光LAMP检测方法及荧光可视化LAMP检测方法。优化后的3种LSDV LAMP方法灵敏度均可达20 copies/反应的靶基因,与WOAH推荐的实时荧光PCR方法(5 copies/反应)相近;特异性良好,与山羊痘病毒(GTPV)、绵羊痘病毒(SPPV)、小反刍兽疫病毒(PPRV)、赤羽病病毒(AKV)、蓝舌病病毒(BTV)及口蹄疫病毒(FMDV)均无交叉反应。其中,实时浊度法操作程序简单,实时荧光法反应快速(25 min内完成检测),荧光可视化法不依赖专用仪器及中心实验室。将所建方法应用于临床样本的检测,显示这3种方法均可特异性检测到LSDV,与实时荧光PCR方法检测结果一致性为100%,优于WOAH推荐的普通PCR方法。所建方法具有快速、特异、灵敏、操作简便且不依赖专用仪器等优点,可为LSDV的临床快速筛查提供技术支持。

The goal of this research is to develop a fast and easy-to-operate LAMP detection method for lumpy skin disease virus(LSDV)to monitor LSDV.Multiple sets of LAMP primers were initially designed based on the conserved sequences of LSDV.A set of primers with the highest reaction efficiency was finally determined,which was located within the LSDV095 gene range.Meanwhile,LSDV plasmid kept in our laboratory were developed to offer positive material for molecular biological detection.After a series of optimizations of the reaction,a real-time turbidimetric LAMP assay,a real-time fluorescent LAMP assay and a fluorescence visual LAMP were established,respectively.After the optimization of the reaction system and reaction temperature,the analytical sensitivity of this method was about 20 copies/reaction of LSDV DNA,which was similar to the qPCR method(5 copies/reaction).The LSDV LAMP method could not amplify templates of GTPV,SPPV,PPRV,AKV,BTV or FMDV,demonstrating high specificity.The LAMP method was utilized exclusively to detect LSDV in clinical samples,and the coincidence rates of the results for LAMP and qPCR were 100%.The results suggest that the LAMP detection method is fast,specific,sensitive,and simple without depending on the expensive equipment,which makes it an effective approach for the rapid detection of LSDV in animal products and clinical samples.