Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA

被引:6
作者
Hara-Kudo, Y
Kasuga, Y
Kiuchi, A
Horisaka, T
Kawasumi, T
Kumagai, S
机构
[1] Natl Inst Hlth Sci, Dept Microbiol, Setagaya Ku, Tokyo 1588501, Japan
[2] Azabu Univ, Sch Vet Med, Dept Vet Microbiol, Sagamihara, Kanagawa 2298501, Japan
[3] Japan Womens Univ, Bunkyo Ku, Tokyo 1128681, Japan
[4] Univ Tokyo, Grad Sch Agr & Life Sci, Bunkyo Ku, Tokyo, Japan
关键词
D O I
10.4315/0362-028X-66.9.1675
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.
引用
收藏
页码:1675 / 1680
页数:6
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