摘要
通过对施马伦贝格病毒(Schmallenberg virus,SBV)及其同属各病毒基因序列进行比对分析,设计了针对SBV S基因节段的一组6条特异性引物,经优化建立了SBV RT-LAMP检测方法,可在63℃恒温条件下,于50min内完成目的序列扩增,通过染料法和琼脂糖凝胶电泳法观察结果,其灵敏度可达10TCID50,与荧光RT-PCR方法具有相同的敏感性。特异性试验显示,本方法与同属的沙门达病毒(Shamonda virus)会发生交叉反应,需借助沙门达病毒S基因节段上的一个特异性酶切位点Afl II进行酶切,实现两者的鉴别诊断。对103份临床样品的检测结果显示,本方法与荧光RT-PCR方法符合率为100%。本研究建立的RT-LAMP方法快速、简单、灵敏度高,不仅适用于实验室鉴定,还可应用于口岸、野外等的临床大规模监测。
Based on sequence analysis of Schmallenberg virus（SBV）,a set of 6 LAMP primers were designed targeting the SBV gene S,and an RT-LAMP assay with one step amplifi cation was established after optimization and the assay was fi nished within 50 minutes at 63℃. The results were observed by color change and agarose gel electrophoresis. The detection limit was 10TCID50,almost as sensitive as the real-time RT-PCR. Considering a cross reaction between SBV and Shamonda virus,a unique Afl II restriction site in Shamonda virus was used in the assay and SBV could be distinguished from Shamonda virus. Test of 103 clinical samples showed that the assay was 100% correlated with real-time RT-PCR. The established RT-LAMP was a rapid,simple and sensitive assay,suitable for use both in laboratory test and clinical fi eld surveillance.
出处
《中国动物检疫》
CAS
2015年第3期73-77,82,共6页
China Animal Health Inspection
基金
质检公益性行业专项(201210018)
国家科技支撑计划课题(2013BAD12B00)
