應用具有內增幅控制之多套式聚合酵素鏈鎖反應於牡蠣中偵測致病性之霍亂弧菌、腸炎弧菌與創傷弧菌   全文下載

Detection of Toxigenic Vibrio cholerae, V. parahaemolyticus and V. vulnificus in Oyster by Multiplex-PCR with Internal Amplification Control

本研究發展具有內增幅控制（IAC）的多套式聚合酵素鏈鎖反應用以同時偵測三種帶有致病基因的弧菌（創傷弧菌的vvhA 與vvp, 腸炎弧菌的tdh, 霍亂弧菌的ctx）。在本實驗中IAC製作為一個嵌合式核酸，綠色螢光基因（gfp）兩端為增幅vvhA 引子序列，在這些弧菌中，增幅片段大小為：IAC (753 bp), vvhA (505 bp), vvp (383 bp), tdh (256 bp) 和 ctx (167 bp)。我們使用了28 株創傷弧菌、18株腸炎弧菌、14株霍亂弧菌和40 株其他弧菌與非弧菌菌株分析中證明本方法的準確性，Kappa index為0.96，在牡蠣均質樣品中，經過8小時的增菌後，可以偵測低至101 CFU/mL 添加於樣品中的弧菌。本方法所應用新製作的IAC和新設計的ctx引子，可以用來快速偵測海產中常見的致病性弧菌。"

This study developed a multiplex-polymerase chain reaction (m-PCR) method that targets the toxin genes, vvhA and vvp for Vibrio vulnificus, tdh for V. parahaemolyticus, ctx for V. cholera, to detect these three species simultaneously. A chimeric DNA consisting of a fragment of the green fluorescent protein gene (gfp) flanked by sequences of the vvhA primers was used as the internal amplification control (IAC). In the presence of these three Vibrio species, amplicons of IAC (753 bp), vvhA (505 bp), vvp (383 bp), tdh (256 bp) and ctx (167 bp) could be simultaneously detected. The accuracy of the m-PCR approach was evaluated with a Kappa index of 0.96 using 28 strains of V. vulnificus, 18 V. parahaemolyticus, 14 V. cholerae and 40 other Vibrio species and non-Vibrio strains. As little as 101 CFU/mL of these Vibrio species in spiked oyster homogenate could be detected by this m-PCR method following enrichment at 37oC for 8 h. This method can be adopted for the rapid detection of the toxigenic strains of Vibrio species in seafood.