实时荧光多酶恒温扩增技术检测小鼠细小病毒核酸的方法建立

Development of a real-time fluorescence-based multienzyme isothermal rapid amplification for the rapid detection of minute virus of mice

目的旨在建立基于实时荧光多酶恒温扩增(exo-MIRA)技术的小鼠细小病毒(MVM)核酸检测新方法。方法收集2021年1月至2022年1月南京大学医学院附属金陵医院实验动物室共75份小鼠肠道内容物标本。针对MVM VP2基因的保守区域设计4对引物,琼脂糖凝胶电泳分析扩增产物以筛选最优引物对,并设计特异性荧光探针,构建exo-MIRA检测法。通过检测梯度稀释模拟样本,进行检出限评价。检测其他小鼠病毒,包括仙台病毒(SV)、小鼠肝炎病毒(MHV)、小鼠鼠痘病毒(Ect)、小鼠肺炎病毒(PVM)、呼肠孤病毒(Reo),分析exo-MIRA法的交叉反应性。采用qPCR法和exo-MIRA法同时检测收集的75份标本,统计分析两种方法的一致性。以qPCR法为参比方法,评估该法的敏感度和特异度,进行诊断效能评价。结果采用exo-MIRA技术建立MVM核酸检测方法,该方法检测MVM的检出限为10 copies/μL,且与其他五种小鼠病毒无交叉反应,可特异性检出MVM。75份标本检出结果显示,exo-MIRA法和qPCR法高度一致,Kappa值为0.921(P<0.05),exo-MIRA法的敏感度为100.0%,特异度为96.7%。结论建立了一种基于实时荧光MIRA恒温扩增技术的MVM核酸检测方法,具有简单快速、实验要求低、敏感特异等优点。该方法可应用于现场复杂环境的即时检测,在实验动物质量检测应用上有一定前景。

Objective To develop a real-timemultienzyme isothermal rapid amplification(MIRA)assay for detecting minute virus of mice.Methods A total of 75 intestinal contents of the mice were collected from January 2021 to January 2022 at the Department of Laboratory Animal,Jinling Hospital.Four pairs of primers were designed to target the conserved region of the MVM VP2 gene.The amplification products were analyzed by gel electrophoresis to select the best primers.Then,specific probes were designed to develop the exo-MIRA assay.To verify the limit of detection of the method,10-fold gradient dilution templates were selected by the exo-MIRA assay.To evaluate the cross-reactivity,viral pathogens were detected by the exo-MIRA,including sendai virus,mouse hepatitis virus,ectromelia virus,pneumonia virus of mice and reovirus.All specimens were run simultaneously by both qPCR and exo-MIRA and the two assays were compared for congruence.The qPCR was used as the reference method.Diagnostic evaluation was performed.Results This study used the exo-MIRA technology to establish a novel assay for minute virus of mice nucleic acid.The limit of detection of the assay was 10 copies/μL and no cross reaction was found with other five mice viruses tested.The detection resulted of clinical specimens identified by the two methods were highly consistent(Kappa=0.921,P<0.05).The exo-MIRA assay achieved a sensitivity of 100.0%and a specificity of 96.7%.Conclusion A real-time multienzyme isothermal rapid amplification assay based on the VP2 gene is developed for the rapid detection of minute virus of mice.