[Detection of Vibrio parahaemolyticus by PCR method with internal amplification control].
OBJECTIVE
The aim of the study was to 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 x 10(2) 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.


CONCLUSIONS
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.
He
Xiaohua
X
Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China. hexiehua123456@sjtu.edu.cn
Yui
Shuijing
S
Chen
Wanyi
W
Shi
Chunlei
C
Meng
Jianghong
J
Shi
Xianming
X
D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D005516:Food Microbiology; D016133:Polymerase Chain Reaction; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification*
D017931:DNA Primers