Real-time PCR快速定量純菌與牡蠣之Vibrio parahaemolyticus菌數

Rapid Quantification of Vibrio parahaemolyticus in Pure Culture and Oyster by Real-time PCR

鄭景陽(Ching-Yang Cheng) ； 黃个晏(Tin-Yan Huang) ； 紀璟叡(Jing-Ruei Ji) ； 林欣榮(Sin-Rong Lin) ； 仇志強(Chi-Chiang Chou) ； 黃錦城(Chin-Cheng Huang)

《臺灣農業化學與食品科學》 45卷1期 (2007/02) Pp. 61-66

https://doi.org/10.6578/TJACFS.2007.008

摘要

以即時聚合酶連鎖反應(real-time polymerase chain reaction; real-time PCR)針對腸炎弧菌(Vibrio parahaemolyticus)之致病基因tlh作為專一性的引子和探針為標的，快速定量純菌腸炎弧菌之菌數，並與其典型菌落之thiosulphate-citrate-bile salts-sucrose(TCBS)平板培養法比較，結果顯示腸炎弧菌以real-time PCR檢測結果和所培養的菌數之相關係數可達0.98。定量腸炎弧菌之DNA量的靈敏度為2×10^0 copies。將已知菌數的腸炎弧菌接種至牡蠣肉中，然後萃取菌體DNA，以real-time PCR法定量腸炎弧菌之靈敏度約1.32log CFU/g。包括樣品之抽取與純化，總共檢測時間只要12小時，因此可應用在牡蠣腸炎弧菌之快速檢測。

關鍵字

即時聚合酶連鎖反應 ； 聚合酶連鎖反應 ； 快速檢測法 ； 腸炎弧菌

並列摘要

Real-time PCR method with primers and probe specific to tlh gene was used as a rapid approach to quantitatively determine pathogenic Vibrio parahaemolyticus species. The result showed that the correlation coefficient between real-time PCR estimates and thiosulphate-citrate-bile salts-sucrose (TCBS) plate counts of V. parahaemolyticus was 0.98. The sensitivity of real-time quantitative PCR assay was 2×10^0 copies for pure V. parahaemolyticus culture. A known number of V. parahaemolyticus target cells were inoculated to oyster and DNA was extracted for real-time PCR analysis. The sensitivity was 1.32log CFU/g for V. parahaemolyticus inoculated to the oyster. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12h. This study demonstrated that real-time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tlh gene in pure culture and in oyster.

並列關鍵字

Real-time PCR ； Polymerase chain reaction ； Rapid detection method ； Vibrio parahaemolyticus