DETECTION OF ADENOVIRUSES AND ENTEROVIRUSES IN POLLUTED WATERS BY NESTED PCR AMPLIFICATION
DETECTION OF ADENOVIRUSES AND ENTEROVIRUSES IN POLLUTED WATERS BY NESTED PCR AMPLIFICATION
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DOI：
10.1128/aem.60.8.2963-2970.1994
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发表时间：
1994-08-01
影响因子：
4.4
通讯作者：
GIRONES, R
中科院分区：
文献类型：
--
作者：
PUIG, M;JOFRE, J;GIRONES, R
建立了一种快速检测环境样品中肠道病毒和腺病毒的方法。通过向不同的污水样品中加入2型腺病毒和1型脊髓灰质炎病毒，测试了几种病毒浓缩和核酸提取系统。最有希望的病毒回收方法包括通过离心浓缩病毒，并通过用0.25 N甘氨酸缓冲液（pH 9.5）处理洗脱病毒沉淀。通过将RNA和DNA吸附到二氧化硅颗粒上的核酸提取是最有效的。一份提取的核酸用于巢式两步PCR，使用所有腺病毒的特异性引物;另一份用于合成cDNA用于巢式两步PCR，使用特异性引物进一步检测河水和污水样品中的接种脊髓灰质炎病毒或所有肠道病毒。评价了该引物的特异性和敏感性，并对24种不同的肠道病毒株和47种人腺病毒血清型进行了识别。估计每个供试种属的灵敏度为1 - 10个病毒颗粒。对25份污水和污染河水样品进行了分析，结果表明巢式PCR法检出的阳性菌株数远高于组织培养法。基于PCR的肠道病毒和腺病毒的检测显示出良好的效果，作为可能的病毒污染的环境废水中的指示。
A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river mater were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.