依赖核酸扩增技术在轮状病毒检测中的应用

Application of nucleic acid sequence-based amplification in the detection of rotavirus

目的将一种新颖的核酸扩增技术—依赖核酸扩增技术(nucleic acid sequence-based amplification NASBA)应用于轮状病毒的快速检测与辅助诊断。方法根据美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)轮状病毒VP7基因保守序列设计引物,建立NASBA检测方法,进行特异性、灵敏度和重复性试验,以所建立方法和RT-PCR方法同时对108例临床病毒性腹泻患者粪便样本进行检测。结果所建立的NASBA方法对轮状病毒具有较好的稳定性和高度特异性,与诺如病毒等8种腹泻相关病毒均无交叉反应,灵敏度达5μg/L。108份粪便样本中,用RT-PCR方法检出轮状病毒阳性11份,用NASBA方法检出阳性样本13份。结论本研究应用的轮状病毒NASBA检测方法特异、灵敏、快速简便,适用于轮状病毒的日常监测和暴发疫情的应急诊断。

Objective To assess the application of nucleic acid sequence-based amplification （NASBA） in the detection of rotavirus. Methods Based on the conservative region of VP7 gene of rotavirus published in Gen- Bank, a pair of primers was designed for NASBA assay. The sensitivity, repeatability and specificity of the assay were evaluated on fecal samples from 108 patients with viral diarrhea. Results NASBA assay could detect rotavirus with high specificity, sensitivity and stability, and showed no cross reactions with 8 other diarrhea-associated virus such as norovirus GI/G H. Out of 108 fecal specimens, 13 were found positive for rotavirus RNA with NASBA method, while only 11 were positive with RT-PCR. Conclusions The new NASBA method is fast, simple, sensitive and specific in the detection of rotavirus and is suitable for rotavirus regular surveillance and rapid diagnosis during disease outbreak.