罗斯河病毒RT-LAMP检测方法的建立

Development of a RT-LAMP Assay for Detection of Ross River Virus

根据Gen Bank中收录的罗斯河病毒(RRV)基因序列,以其保守的E2基因为靶序列,设计并筛选出一套特异性LAMP引物;通过优化反应条件,最终建立了RRV RT-LAMP检测方法,并对其灵敏度、特异性进行了验证。结果显示:本方法对RNA样本的最低检测限可达10拷贝/25μL,而荧光RT-PCR方法和常规RT-PCR方法的灵敏度分别为10拷贝/25μL、100拷贝/25μL;其特异性良好,不与同属或相似病毒发生交叉反应。结果表明,本方法灵敏、特异、快速、便捷,可应用于我国虫媒病高发区的RRV筛查预警。

According to the gene sequences of Ross River virus(RRV)published in GenBank,a set of specific LAMP primers were designed and screened by taking the conserved E2gene as the template.After optimizing the reaction conditions,a RT-LAMP assay for RRV detection was developed,and its sensitivity and specificity were evaluated.According to the results,the detection limit of RNA was10copies/25μL,while those of real-time RT-PCR method and conventional RT-PCR method were found to be10and100copies/25μL,respectively.The specificity was good,and there was no cross reaction between the assay with the same genus or similar virus.As a result,the developed assay was sensitive,specific,rapid and convenient,and it could be applied in RRV screening and early warning in high incidence areas of insect-borne diseases.