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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 apply a novel nucleic acid amplification-based nucleic acid sequence-based amplification (NASBA) technique to the rapid detection and diagnosis of rotavirus. Methods According to the conservative sequence of VP7 gene of the National Center for Biotechnology Information (NCBI), a NASBA assay was designed to test the specificity, sensitivity and reproducibility. The established method and RT-PCR Methods At the same time, 108 cases of clinical viral diarrhea patients stool samples were tested. Results The established NASBA method showed good stability and high specificity for rotavirus. No cross-reaction was found with 8 kinds of diarrhea-related viruses such as norovirus and the sensitivity was 5μg / L. In 108 stool samples, 11 rotavirus positive samples were detected by RT-PCR and 13 by NASBA. Conclusion The rotavirus NASBA detection method used in this study is specific, sensitive, rapid and simple, and is suitable for routine surveillance of rotavirus and emergency diagnosis of outbreaks.