Kim Duwoon, Kim Seok-Ryel, Kwon Ki-Sung, Lee Ji-Won, Oh Myung-Joo
Division of Food Science and Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea.
J Microbiol. 2008 Aug;46(4):436-40. doi: 10.1007/s12275-008-0131-1. Epub 2008 Aug 31.
The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 10(5) fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.
利用聚合酶链反应的分子方法已被提议作为鉴定食品和水中病毒病原体的有用工具。然而，由于在存在PCR抑制剂的情况下PCR灵敏度较低，基于PCR的方法高度依赖于病毒浓缩和核酸纯化方法。我们开发了TPTT [三羟甲基氨基甲烷洗脱缓冲液-聚乙二醇-TRIzol-聚（dT）磁珠]方案，以检测接种在牡蛎消化腺中的甲型肝炎病毒（HAV）。在用氢氧化锆沉淀的HAV中，巢式PCR的检测限比用PEG/NaCl（16%聚乙二醇6000，0.525 M NaCl）以1:2（v/v）比例两次沉淀HAV上清液的检测限低10^5倍，后者能从约0.05 g牡蛎匀浆中高效检测出0.0148 PFU/g。该方法在检测贝类中的HAV方面具有潜在的高效性，且比目前大多数已发表的检测方法更灵敏。
