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| Simultaneous Detection of Vibrio parahaemolyticus and V .cholerae from Aquatic Animals by Dulplex PCR |
| ZHANG Xiao-jun,BAI Xue-song,BI Ke-ran,ZHOU Bao-yun,QIN Lei,YAN Bin-lun,QIN Guo-min |
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Abstract V ibrio parahaemolyticus and V . cholerae were known to be responsible not only for aquatic animals ,but also to be recognized as pathogenic bacteria for human .In this study ,two pairs of specific primers were designed based on toxR gene for V . parahaemolyticus and lolB gene for V .cholerae ,and simultaneous detection of the both V ibrio was established by a dulplex polymerase chain reaction (PCR) , amplification of 368 bp and 519 bp gene fragments .The results showed that two purposed gene fragments from mixed chromosomal DNA of V . parahaemolyticus and V . cholerae in one PCR reaction were simultaneously amplified by two PCR primers ,and no positive reaction was detected in 5 other controlled pathogenic bacteria , showing strong specificity , and no interference between the two primers . The sensitivity test of the dulplex PCR revealed that the two primers detected V .parahaemolyticus at a level of as few as 1 .75 × 102 cfu/mL ,and V .cholerae at a level of as few as 1 .25 × 102 cfu/mL .The positive amplified results were observed in the 8 aquatic products infected artificially ,and negative amplifi cation in the no-infected 8 aquatic products . The findings prove that the dulplex PCR is a specific and sensitive method for simultaneous detection of the both V ibrio in one PCR reaction ,and can be used as the detection of food safety ,and diagnose of aquatic animals diseases caused by the both V ibrio .
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Received: 25 July 2013
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