Jean Julie, D'Souza Doris H, Jaykus Lee-Ann
Food Science Department, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, USA.
Appl Environ Microbiol. 2004 Nov;70(11):6603-10. doi: 10.1128/AEM.70.11.6603-6610.2004.
Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.
人类肠道病毒目前被认为是食源性疾病最重要的病因之一。由于现有检测方法的不足，肠道病毒在食源性疾病暴发中的作用可能难以确定。在本研究中，开发了一种基于核酸序列扩增（NASBA）的多重检测方法，用于特异性、同时且快速地检测与流行病学相关的人类肠道病毒。在一个单一反应中使用了三个先前报道的引物组，分别扩增474、371和165个核苷酸的RNA靶片段，用于检测甲型肝炎病毒以及I基因组和II基因组诺如病毒。扩增产物通过琼脂糖凝胶电泳进行检测，并通过电化学发光和Northern杂交进行确认。多重NASBA检测的终点检测灵敏度约为每个反应10^(-1)个逆转录PCR可检测单位（或视情况为PFU）。当用不同浓度的每种病毒接种代表性即食食品（熟食切片火鸡和生菜），并使用多重NASBA方法进行病毒检测时，在两种食品中，初始接种水平为10^(0)至10^(2)个逆转录PCR可检测单位（或PFU）/9平方厘米时，所有三种人类肠道病毒均能同时被检测到。多重NASBA系统可在单个反应管中快速同时检测临床上相关的食源性病毒，对于检测食品中的病毒污染而言，可能是逆转录PCR的一种有前景的替代方法。
