Rapid Detection of Vibrio parahaemolyticusby Real-time PCR using High Resolution Melting Curve Analysis
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Abstract:
A rapid real-time PCR method for detecting Vibrio parahaemolyticus (VP) by high resolution melting curve analysis (HRM) was developed. A pair of specific primers targeting the toxR gene of Vibrio parahaemolyticus was used. After optimizing the reaction system and conditions,the specificity of the method was validated with 10 target strains and 10 non-target strains,Serial dilutions from 3.50×101 to 3.50×107 copies/mL purified constructed plasmids were analyzed to evaluation its sensitivity. Reproducibility assay was also conducted on purified constructed plasmids between 1.50×101 and 1.50×107 copies/mL. The method was applied to detect 90 live submitted seafood samples. The results showed that the developed HRM real-time PCR assay had a good specificity by detecting only VP with the Tm value of 76.64±0.57 ℃ and was not affected by other seafood pathogens such as Vibrio vulnificus, Vibrio cholerae,Staphylococcus aureus et al. The sensitivity of detection of the constructed plasmids were 3.50×102 copies/mL. In addition, the assay had a high reproducibility and the average Tm values were 76.53±0.35 ℃ and 76.74± 0.52 ℃. The sample’s variations (CVs) were 0.56±0.42% and 1.11±0.73%. A better detection rate of 18.89% than that of the traditional culture method (14.44%) was found in the detection of 90 live seafood samples. The established real-time PCR assay showed sufficient specificity, high sensitivity and good reproducibility. And it was rapid, simple and cheap for detection of VP.
