Jean Julie, Blais Burton, Darveau André, Fliss Ismaïl
Département de Sciences des Aliments et de Nutrition, Université Laval, Québec, G1K 7P4, Quebec, Canada.
J Virol Methods. 2002 Aug;105(1):123-32. doi: 10.1016/s0166-0934(02)00096-4.
Human rotavirus and hepatitis A virus (HAV) are two of the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for their detection in foodstuffs. In this study, a multiplex nucleic acid sequence-based amplification (NASBA) system was developed to detect specifically and simultaneously human rotavirus and HAV. Two sets of primers selected from published nucleic acid sequences were used in the NASBA mixture to amplify viral RNA from both viruses. Denaturing gel electrophoresis revealed two distinct RNA products with 268 and 474 nucleotides amplified from rotavirus and HAV, respectively. The specificity of the multiplex NASBA was confirmed by a microtiter plate hybridization and detection system and by Northern blot analysis using specific oligonucleotide probes. The presence of non-homologous nucleic acid and non-target microorganisms did not have any effect on the specificity of the multiplex NASBA. Using the optimized NASBA and microtiter plate hybridization conditions, as little as 400 PFU ml x (-1) of HAV and 40 PFU ml x (-1) of rotavirus were detected. The multiplex NASBA system offers advantages over monoplex virus detection systems in terms of turnaround time and cost-effectiveness.
人类轮状病毒和甲型肝炎病毒（HAV）是病毒介导的食源性疾病最常见的两种病因。与这些病毒相关的疫情流行病学调查一直受到食品中缺乏可用检测方法的阻碍。在本研究中，开发了一种基于多重核酸序列扩增（NASBA）的系统，用于特异性同时检测人类轮状病毒和HAV。从已发表的核酸序列中选择的两组引物用于NASBA混合物中，以扩增两种病毒的病毒RNA。变性凝胶电泳显示分别从轮状病毒和HAV扩增出268和474个核苷酸的两种不同RNA产物。通过微量滴定板杂交和检测系统以及使用特异性寡核苷酸探针的Northern印迹分析证实了多重NASBA的特异性。非同源核酸和非靶标微生物的存在对多重NASBA的特异性没有任何影响。使用优化的NASBA和微量滴定板杂交条件，可检测到低至400 PFU ml×（-1）的HAV和40 PFU ml×（-1）的轮状病毒。多重NASBA系统在周转时间和成本效益方面比单重病毒检测系统具有优势。
