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囚的弧菌（創傷弧菌的vvhA與vvp，腸炎弧菌的tdh，霍亂弧茵的ctx）。在本實驗中IAC製作為一個嵌合式核酸，綠色螢光基因（grp）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。

關鍵字

創傷弧菌；腸炎弧菌；霍亂弧菌；多套式聚合酵素鏈鎖反應；內增幅控制；海產

並列摘要

This study developed a multiplex-polymerase chain react ion (m-PCR) method that targets the toxin genes, vvhA and vvp for Vibrio vulnificus, tdh for V. parahaemolvticus, 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, 18V. parahacmolyticus, 14 V cholcrae and 40 other Vibrio species and non-Vibrio strains. As little as 10^1 CFU/mL of these Vibrio species in spiked oyster homogenate could be detected by this m-PCR method following enrichment at 37℃ for 8 h. This method ca n be adopted for the rapid detection of the toxigenic strains of Vibrio species in seafood.

並列關鍵字

Vibrio vulnificus ； Vibrio cholera ； Vibrio parahaemolyticus ； internal amplification control ； multiplex PCR ； virulence ； oyster

國際替代計量

Detection of Toxigenic Vibrio Cholerae, V. Parahaemolyticus and V. Vulnificus in Oyster by Multiplex-PCR with Internal Amplification Control

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