Real-time PCR For Detection Of Five Species Of Vibrio

Vibrio species are naturally diverse bacteria that inhabit aquatic environments and marine animals.Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus,Vibrio alginolyticus and Vibrio mimicus are major concerns,as they are pathogenic to animals,including humans.It is necessarry to develop a sensitive and fast detection method.V parahaemolyticus is recognised as the leading cause of human gastroenteritis associated with seafood consumption.Thermostable direct hemolysin(TDH)and heat-related hemolysin(TRH)are included in major virulence factors.According to the sequences of toxR gene which is specific between species,virulent genes tdh and trh,three pairs of specific primers and fluorogenic-labedled probes of V.parahaemolyticus were designed,and applied to real-time PCR.By optimizing the reaction system and the parameters,the single and multiplex real-time PCR methods were developed for the identification of V parahaemolyticus.The real-time PCR methods had a high specificity,and no cross-reactivity was observed with other tested bacteria.The sensitivity of the single real-time PCR was 100 times higher than normal PCR,while the multiplex real-time PCR was found to have a lower limit of 103 colony-forming units(CFU)on for V parahaemolyticus.The coefficient of variation in repetitive test was less than 1.8%,indicating good repeatability of the method.According to the sequences of ompW gene specific for V.cholerae,and virulent genes ctx and hly,three pairs of specific primers and fluorogenic-labedled probes were designed,and applied to real-time PCR.By optimizing the reaction system and the parameters,the single and multiplex real-time PCR methods were developed for the identification of V.cholerae.The real-time PCR methods had high specificity,and no cross-reactivity was observed with other tested bacteria.The sensitivity of the single real-time PCR was 1000 times higher than normal PCR,while the multiplex real-time PCR was found to have a lower limit of 102 CFU for V.cholerae.The coefficient of variation in repetitive test was less than 1.1%,indicating good repeatability.According to the sequences of vvhA,vmhA and vacol,which are specific for V vulnificus,V.mimicus and V.alginolyticus,three pairs of specific primers and the fluorogenic-labedled probes were synthized.By optimizing the reaction system and the parameters,a multiplex real-time PCR method was developed for the identification of the three bacteria.The real-time PCR methods had high specificity,and no cross-reactivity was observed with other tested bacteria.The sensitivity of a single real-time PCR response was 100-1000 times higher than normal PCR,while the multiplex real-time PCR was found to have a lower limit of 3×1033?3×104 CFU for the three bacteria The coefficient of variation in repetitive test was less than 1.8%,indicating good repeatability.In conclusion,the real-time PCR methods were developed for the detection of V.cholerae,V.parahaemolyticus,V vulnificus,V.alginolyticus and V.mimicus.This study provides some useful tools for investigating and monitoring the five species of vibrio.