Xiaohua He
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaShuijing Yu
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaWanyi Chen
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaChunlei Shi
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaJianghong Meng
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaXianming Shi
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center,School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaSupported by Grants 09DZ0503300，091422021200，08DZ0504200 from the Science and Technology Commission of Shanghai Municipality; and the special grant 2009BAK43B31 for Expo from the Ministry of Science and Technology of the People’s Republic of China
Abstract: [Objective] The aim of the study was explore a new detective target in Vibrio parahaemolyticus which was more specific than others at present, to construct an internal amplification control (IAC), and finally to develop a new PCR system which could effectively eliminate false-negative results. [Methods] Genomic comparison analysis was used to explore V. parahaemolyticus specific targets, which were then evaluated and used to design specific primers. An IAC was constructed by the compound primer technology. PCR parameters were optimized, and its reaction system was developed. [Results] A V. parahaemolyticus species-specific sequence (vp1332) which encodes probable binding protein component of ABC transporter was mined and selected as a detection target. A pair of specific primers (vp1332L/vp1332R) was designed based on this sequence for the development of a PCR assay. An internal amplification control was constructed and added into the PCR detection system, which was co-amplified with the target sequence so as to indicate the existence of inhibitors. Specificity of this PCR system was tested with 296 V. parahaemolyticus strains and 33 non- V. parahaemolyticus strains, and the results showed that there was a 343-bp amplicon resulted from all V. parahaemolyticus strains, while there was no this amplicon but only a 499-bp IAC amplicon appeared for all non- V. parahaemolyticus strains. The detection limit of this assay for purified V. parahaemolyticus genomic DNA was 1.6×102 cfu/mL. Artificial contamination assays showed that V. parahaemolyticus could be detected after eight hours enrichment when the original concentration of this bacterium was 1.24 cfu/25 g. The PCR method developed in this study was also evaluated with Seafood samples, and the results demonstrated that it worked effectively. [Conclusion] A novel PCR method was successfully developed in this study, which could effectively detect V. parahaemolyticus with high accuracy and could especially eliminate false-negative results.
Xiaohua He, Shuijing Yu, Wanyi Chen, Chunlei Shi, Jianghong Meng, Xianming Shi. Development of a PCR method with an internal amplification control for the detection of Vibrio parahaemolyticus. [J]. Acta Microbiologica Sinica, 2010, 50(3): 387-394
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