Jaykus L A, De Leon R, Sobsey M D
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill 27599-7400, USA.
Appl Environ Microbiol. 1996 Jun;62(6):2074-80. doi: 10.1128/aem.62.6.2074-2080.1996.
This article reports the development of a method to purify and concentrate intact virions from oyster extracts to volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridization. Fifty-gram oyster samples were processed by an adsorption-elution -precipitation method and then seeded with 10(1) to 10(5) PFU of poliovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oyster extracts were purified by fluorocarbon extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Virus recovery after elution of PEG precipitates was dependent upon PEG concentration and averaged 60% for PV1 and 40% for HAV. The next processing step used the protein-precipitating agent Pro-Cipitate (Affinity Technology, Inc., Brunswick, N.J.) in an adsorption-elution -precipitation scheme to further concentrate viruses and reduce sample volumes to 100 microliter. Oyster extracts processed by Pro-Cipitate adsorption-elution-precipitation were directly compatible with RT-PCR and yielded virus recoveries of > 80% for both PV1 and HAV. When extracts from 50-g oyster samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10 PFU for both PV1 and HAV and with low levels of Norwalk virus. Virus recoveries based on cell culture infectivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on artificially contaminated raw oysters, the combined method successfully detected > or = 10(3) PFU of PV1 and HAV and 10(5) RT-PCR-amplifiable units of Norwalk virus. Virus detection by RT-PCR and cell culture infectivity was consistent and well correlated among replicate samples and at different virus titers. The procedure developed in this study is rapid, sensitive, and effective for the direct detection of enteric viruses in oysters by RT-PCR.
本文报道了一种从牡蛎提取物中纯化和浓缩完整病毒粒子的方法，该方法所获病毒粒子的量和质与通过逆转录聚合酶链反应（RT-PCR）进行病毒基因组核酸扩增以及通过寡核苷酸探针杂交进行确认相匹配。对50克牡蛎样本采用吸附 - 洗脱 - 沉淀法进行处理，然后接种1型脊髓灰质炎病毒（PV1）或甲型肝炎病毒（HAV）的10(1)至10(5)空斑形成单位（PFU）。牡蛎提取物中的接种病毒通过氟碳萃取进行纯化，并通过聚乙二醇（PEG）沉淀和洗脱进行浓缩。PEG沉淀洗脱后的病毒回收率取决于PEG浓度，PV1平均为60%，HAV平均为40%。下一步处理步骤在吸附 - 洗脱 - 沉淀方案中使用蛋白质沉淀剂Pro-Cipitate（Affinity Technology, Inc., Brunswick, N.J.）进一步浓缩病毒，并将样本体积减少至100微升。经Pro-Cipitate吸附 - 洗脱 - 沉淀处理的牡蛎提取物与RT-PCR直接兼容，PV1和HAV的病毒回收率均>80%。当对50克牡蛎样本的提取物进行接种并通过联合浓缩和纯化方案处理时，对于PV1和HAV以及低水平的诺如病毒，在初始接种量为10 PFU时即可直接通过RT-PCR检测病毒基因组RNA。基于细胞培养感染性的病毒回收率，PV1为25%至35%，HAV为5%至10%。在人工污染的生牡蛎上进行测试时，该联合方法成功检测到PV1和HAV的>或 = 10(3) PFU以及诺如病毒的10(5)个RT-PCR可扩增单位。RT-PCR检测病毒和细胞培养感染性在重复样本以及不同病毒滴度之间是一致且相关性良好的。本研究中开发的程序对于通过RT-PCR直接检测牡蛎中的肠道病毒而言快速、灵敏且有效。
