The Use of Polymerase Chain Reaction for Rapid Detection of Vibrio parahaemolyticus

• Main Authors: Pan,Shwu-Fen, 潘淑芬
• Other Authors: Chiayin Lee
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/98323334256719825275

碩士 === 國立臺灣大學 === 農業化學系 === 81 === A rapid polymerase chain reaction (PCR) method was developed for detection of Vibrio parahaemolyticus in clinical and food samples, respectively. We used a pair of primers VP21 、VP22 based on tdh gene to detect V. parahaemolyticus which had tdh gene. VP21、VP22 were evaluated with 175 isolated microorganisms including 40 V. parahaemolyticus which had tdh gene, 84 V. parahaemolyticus which didn't possess tdh gene, and 51 other Vibrios and enteric pathogens. Amplification products were confirmed by southern blot hybridization with 32P-VP5 probe. The data shown only 40 V. parahaemolyticus which had tdh gene can be amplified in PCR and VP21、VP22 had a specificity of 100%. In the analysis of 27 stool samples, V. parahaemolyticus was isolated by conventional methods from 14 stool samples, but TDH- producing V. parahaemolyticus was found in 13 of 14. With PCR, 12 stool samples can be amplified. The detection rate of TDH-producing V. parahaemolyticus in stool samples was 92.3%. Recently, several V. parahaemolyticus isolates from clinical samples were found to produce a TDH-related hemolysin (TRH). A survey of the presence of the trh gene in clinical and environ- mental strains of V. parahaemolyticus provided information on the significance of TRH as a virulence factor. So, the development of a PCR method to detect V. parahaemolyticus in food samples was needed. We used a pair of primer VP33、VP32 based on UP fragment to detect V. parahaemolyticus, and confirmed by southern blot and dot blot hybridization with 32P-VP35. In specific test, correct PCR products were obtained in all V. parahaemolyticus (124/124) and 1 V. alginolyticus. No PCR products were found in other 50 non-V. parahaemolyticus. Furthermore, a PCR procedure which allows the direct detection of V. parahaemolyticus in oyster was developed. The heated oyster was artificationlly contaminated with various