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目的建立能同时检测水环境中肠道病毒(Enterovirus,EVs)、诺如病毒GⅡ(Norovorus,Nov GⅡ)、轮状病毒(Rotavirus,RVs)、腺病毒(Adenovirus,AdVs)的多重RT-PCR检测方法。方法根据GenBank收录的肠道食源性病毒核酸序列,利用软件DNAMAN设计肠道病毒通用引物以及Nov GⅡ、RVs、AdVs的特异性引物;将这4对引物置于同一体系中进行多重RT-PCR,利用各种肠道食源性病毒的核酸模板检测其特异性并通过不同滴度[100～107pfu(copies)/ml]病毒测试其灵敏度;利用加标实验验证该方法的准确性;通过对5份河水或某水厂出水大体积水样(50 L)进行病毒富集与检测,以确定该方法的实用性,同时,采用传统细胞培养法验证其结果。结果该方法对目的病毒均获得了理想的电泳条带,而对其他病毒无非特异扩增;对EVs、AdVs的最低检出滴度均为10 pfu/ml,Nov GⅡ-4为10 copies/ml,对RVs的最低检出滴度为102pfu/ml;加标实验检测结果准确率为100%;对5份河水或某水厂出水大体积水样进行病毒富集与检测,对以上各种病毒均有检出且与细胞培养结果一致。结论该方法不仅特异性强、灵敏度高、简便快捷,而且准确率高,实际应用性强,适用于水中肠道食源性病毒的快速检测。
Objective To establish a multiplex RT-PCR assay capable of simultaneously detecting Enterovirus (EVs), Norovorus (Nov G Ⅱ), Rotavirus (RVs) and Adenovirus (AdVs) method. Methods According to the sequence of gut-derived viral sequences contained in GenBank, the universal primer of enterovirus and the specific primers of Nov GⅡ, RVs and AdVs were designed by software DNAMAN. The four pairs of primers were placed in the same system for multiplex RT-PCR The specificity of the method was tested by using nucleic acid templates of various enteric viruses and their sensitivity was tested by different titers of [100 ~ 107pfu copies / ml] of virus. The accuracy of the method was verified by using spiking experiments. Five rivers or a water plant effluent with a large volume of water samples (50 L) for virus enrichment and testing to determine the practicality of the method, at the same time, the use of traditional cell culture method to verify the results. Results The method was able to obtain ideal electrophoresis bands for target virus and non-specific amplification for other viruses. The lowest detectable titer of AdVs was 10 pfu / ml for EVs and 10 copies / ml for Nov GII-4 , The lowest detectable titer of RVs was 102pfu / ml; the accuracy of the spiked test was 100%; the water samples collected from 5 rivers or a water plant were collected for virus enrichment and detection. Both were detected and consistent with cell culture results. Conclusion The method is not only specific, sensitive, simple and quick, but also high accuracy, practical application, suitable for rapid detection of foodborne enteric viruses in water.