斜纹夜蛾成虫带核型多角体病毒的巢式PCR检测

Spodoptera litura Nucleopolyhedrovirus(SpltNPV)in Spodoptera litura Adults:Nested PCR Detection

旨在建立一种快速、可靠的斜纹夜蛾成虫带核型多角体病毒(Spodoptera litura Nucleopolyhedrovirus,SpltNPV)的分子检测方法,以克服目前常规PCR技术很难检测到斜纹夜蛾成虫带病毒的缺陷。以SpltNPV的蜕皮甾体尿苷二磷酸葡萄糖转移酶(ecdysteroid UDP-glucosyltransferase,egt)基因为检测对象,基于该基因序列设计了巢式PCR引物,其中一对外侧引物egt-F/R和一对内侧引物egt1-F/R,并研究斜纹夜蛾成虫带毒的巢式PCR检测可靠性。结果表明:基于egt基因的巢式PCR方法可以特异性扩增SpltNPV的DNA,引物egt-F/R的扩增检测最低限为10-7μg/μL;且基于引物egt-F/R的两轮普通PCR无法检测出成虫带毒,而采用巢式PCR方法可有效扩增出目的egt片段,巢式PCR检测方法的灵敏度比两轮普通PCR检测方法提高2个数量级。因此,本研究成功建立了基于SpltNPV的egt基因的巢式PCR检测技术,该方法为斜纹夜蛾成虫带毒、传毒等流行病学研究提供了可靠的技术支撑。

The aim of this study is to establish a rapid and reliable method for molecular detecting of SpltNPV in Spodoptera litura adults,in order to overcome the defect of the conventional PCR technology.In this paper,the ecdysteroid UDP-glucosyltransferase(egt)gene of Spodoptera litura was used as the detection object.Nested PCR primers were designed based on the egt gene sequences,including a pair of outer primers egt-F/R and a pair of inner primers egt1-F/R.And the reliability of nested PCR detection was studied on Spodoptera litura adults.The results showed that the nested PCR method had strong specificity for SpltNPV DNA amplification,and the minimum limit for amplification detection of primer egt-F/R was 10-7μg/μL.The results also indicated that two rounds of conventional PCR amplification could not detect the SpltNPV by the primers of egt-F/R,and the nested PCR method could effectively amplify the target fragment of gene egt.In addition,the sensitivity of the nested PCR method was 2 orders of magnitude higher than that of the two rounds of the conventional PCR detection method.Therefore,the nested PCR method for detecting SpltNPV was established for Spodoptera litura adults in this study.Moreover,the method can provide technical support for the epidemiological study on the infection and transmission of the virus in Spodoptera litura adults.