Gouvea V, Santos N, Timenetsky M do C, Estes M K
Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, DC 20204.
J Virol Methods. 1994 Jul;48(2-3):177-87. doi: 10.1016/0166-0934(94)90117-1.
A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.
一种轮状病毒双链RNA纯化方案被用于从人工污染的贝类中提取诺沃克单链RNA，并设计了一种用于诺沃克病毒的灵敏逆转录-聚合酶链反应检测方法，以鉴定估计20至200个基因组拷贝。该技术包括用异硫氰酸胍进行脱蛋白、将RNA吸附到羟基磷灰石上，以及用十六烷基三甲基溴化铵和乙醇进行连续沉淀。该方案可从牡蛎、蛤和各种食物基质中高回收率地获得不含酶抑制剂的病毒RNA。通过使用从聚合酶基因中选择的引物来复制和扩增诺沃克病毒序列。用限制性内切酶消化扩增产物可确保检测的特异性。这种快速灵敏的检测方法可能会显著改善对食品中不可培养的诺沃克病毒进行常规筛查的前景。
