Dix A B, Jaykus L A
Food Science Department, North Carolina State University, Raleigh 27695-7624, USA.
J Food Prot. 1998 Apr;61(4):458-65. doi: 10.4315/0362-028x-61.4.458.
A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 10(1) to 10(5) PFU to poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variable yielding recoveries as high as 99% for PV1 and 45% for HAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (< 1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam 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(3) PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for pV1 and HAV, respectively, when extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.
一种从牡蛎提取物中提取并浓缩完整人类肠道病毒以用于逆转录聚合酶链反应（RT-PCR）检测的方法被应用于硬壳蛤（Mercenaria mercenaria）。50克蛤样本采用吸附-洗脱-沉淀法进行处理，然后接种10¹至10⁵空斑形成单位（PFU）的脊髓灰质炎病毒1型（PV1）和/或甲型肝炎病毒（HAV）。提取物中的接种病毒通过氟碳（氟利昂）萃取进行纯化，并通过聚乙二醇（PEG）沉淀和洗脱进行浓缩。通过细胞培养感染性测定评估，从PEG沉淀物中回收病毒粒子的效率取决于PEG浓度和洗脱缓冲液体积，优化后的变量对PV1的回收率高达99%，对HAV的回收率为45%。为了进一步浓缩病毒、去除抑制剂并减少样本体积，在吸附-洗脱-沉淀方案中使用了蛋白质沉淀剂Pro-Cipitate。最终浓缩物体积小（<1毫升），可直接用于RT-PCR进行病毒基因组扩增。当对50克蛤样本的提取物进行接种并采用联合浓缩和纯化方案处理时，对于PV1和HAV，在初始接种水平为10³ PFU时即可直接通过RT-PCR检测病毒基因组RNA。当蛤提取物被诺如病毒人工污染时，基于细胞培养感染性的相应病毒回收率对于PV1和HAV分别为7%至50%和0.3%至8%，使用RT-PCR直接检测病毒粒子RNA并随后进行寡核苷酸探针杂交，在每50克蛤样本提取物中低至450个诺如病毒RT-PCR可扩增单位的水平下即可实现。
