| Vibrio parahaemolyticus is a well-recognized human pathogen, which is widely distributed in marine and estuarine environments. This organism can cause gastroenteritis through ingestion of raw or partially cooked seafood. More attention has been paid to the increasing reports of disease outbreaks in humans, and the accurate identification and rapid detection of this organism is of great importance.PCR technology has been applied widely in the detection of foodborne pathogen for its high sensitivity, specificity and rapidness, and has a wide application foreground. However, the existing detection targets from previous researches are not good enough in quantification and specification and tend to generate false-positive results sometimes. Additionally, the inhibitors would also interference with the PCR amplification to generate false-negative detection results. In order to avoid the false detection results and increase the accuracy in PCR detection of V. parahaemolyticus, the following work was carried out in this study.A target was obtained from 61 candidates by bioinformatics ananlysis, then a pair of primers (vp1332L/R) was designed according this target, based on which a traditional PCR system and a real-time PCR system were developed for detecting the V. parahaemolyticus. The traditional PCR detection system with an IAC for foodborne V. parahaemolyticus was evaluated for its specificity, sensitivity, anti-interference ability and accuracy:1) An IAC was constructed using composite primer method, which was heterologous to the target gene;2) Specificity of this PCR system was tested with 309 V. parahaemolyticus strains and 81 non- V. parahaemolyticus strains, and it was shown that a 343 bp amplicon resulted from all V. parahaemolyticus strains, while only a 499 bp IAC amplicon appeared for all non- V. parahaemolyticus strains;3) Sensitivity of this PCR system was 30.0 fg/μL for the pure genomic DNA (1.6×102 cfu/mL for pure culture) when the concentration of IAC was 4.29×103 copies/PCR, and the detection limit for artificially contaminated seafood samples was 1.24 cfu/25 g if they were cultivated for 8 h in buffered peptone water after contamination;4) Anti-interference ability of this PCR system was evaluated in the presence of 21.5 ng genomic DNA or after 12 h enrichment with contamination of non- V. parahaemotyticus bacteria at 109 cfu/ mL as background, and it was shown that the positive results for the target organism were achieved in either situation;5) Accuracy of this PCR system was evaluated with 106 seafood samples. The results demonstrated that the system with IAC could successfully eliminate the false-negative.The real-time PCR detection system with an IAC for V. parahaemolyticus was evaluated for specificity, sensitivity, anti-interference ability and accuracy: 1) An IAC was constructed using composite primer method and then a probe was designed according to this IAC;2) The specificity of this assay was evaluated by 390 bacterial strains. Only V. parahaemolyticus strains generated a target fluorescent signal, while the non- V. paraheamolyticus strains only generated IAC fluorescent signal;3) When 94 copies of IAC per reaction were used, they had no influence on the detection limit of this assay. The detection limit of this assay was 4.8 fg purified genomic DNA or about 1 genome copy per reaction for V. parahaemolyticus;4) Positive results were also obtained by this assay in the presence of 2.1μg genomic DNA or 107 cfu/mL cells of non- V. parahaemotyticus as background;5) Ninety-six seafood samples were tested with this new developed real-time PCR, and it was found that this real-time PCR assay is effective for the rapid detection of V. parahaemotyticus in the contaminated samples.In conclusion, the IAC-PCR detection system established in this study could successfully eliminate the false-negative, moreover, the whole test was rapid, accurate, specific, and sensitive, which could be a robust alternative for the legitimate detection criterion in V. parahaemolyticus detection.
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