Lee Chi-Ying, Panicker Gitika, Bej Asim K
Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170, USA.
J Microbiol Methods. 2003 May;53(2):199-209. doi: 10.1016/s0167-7012(03)00032-0.
Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.
与食用生贝类尤其是牡蛎相关的疾病爆发是海鲜行业和公共卫生机构主要关注的问题。优化了靶向基因片段的多重PCR扩增，随后进行DNA-DNA夹心杂交以检测病原体。首先，开发了hns、spvB、vvh、ctx和tl的多重PCR扩增，能够同时从纯培养物和接种的牡蛎中检测肠炎沙门氏菌鼠伤寒血清型、创伤弧菌、霍乱弧菌和副溶血性弧菌。然后，使用磷酸化和生物素化的寡核苷酸探针，对扩增产物进行比色CovaLink NH微孔板夹心杂交，其核苷酸序列位于扩增DNA的内部。与多重PCR扩增DNA杂交的结果在405nm波长下显示出14.1至43.2之间的高信噪比。每种病原体的检测灵敏度为10(2)个细胞/g牡蛎组织匀浆。本研究结果表明，多重PCR与比色微孔板夹心杂交测定相结合，可为检测贝类中的微生物病原体提供一个特异、灵敏且可重复的系统，从而提高贝类对消费者的微生物安全性。
