Rapid Detection Of Vibrio Parahemolyticus In Seafood Using The PCR-ELISA Method

In this study,a PCR-ELISA method was established to detect Vibrio parahaemolyticus in seafood and the target were toxR gene and tdh gene.During the process of this study,standard Vibrio parahaemolyticus was used as the research goal.DNA,the PCR template,was extracted from Vibrio parahaemolyticus,two pairs of primers were designed by toxR and tdh gene,then,its specificity was tested.To these two pairs of primers,each pair was labeled,one was labeled with biotin,the other with digoxigenin,so as to the other pair.The labled primers were used in PCR reaction with the programme parameter consisted of an activation step at 94℃ for 3min followed by 30 cycles at 94℃ for 30 s,an annealing temperature at 53℃ for 30 s and an extension at 72℃ for 1min with a final extension step at 72℃ for 10 min.So,biotin and digoxigenin were labeled to PCR production, and PCR production could be connected to the 96-well plate coated with streptavidin.Antidigoxigenin-detecting antibody Fab fragments was added to the well and reacted with digoxigenin,when they reacted fully,then put ABTS in,its color changed and tested the absorbance values of the plates at a wavelength of 405 nm using the ELISA reader.Results were judged by the OD405 values.The number of bacteria in initial bacterial liquid of Vibrio parahaemolyticus was tested by colony counting method and its number was1.03×108 CFU/mL.1 ml bacterial liquid was taken to extract DNA,and the DNA solution was diluted at a 10 fold dilution which were detected by the method established in this study to determine the limit.Finally,the sensitivity was identified as 1.03×102CFU/mL.30 clam samples were collected at the markets in DaLian,Vibrio parahaemolyticus of each sample were detected by GB method(GB 4789.7-2013)and PCR-ELISA method and SYBR GreenⅠfluorescence quantitative PCR method evaluated in this artical.There were 4positive samples were detected by using the PCR-ELISA method which was the same with SYBR Green Ⅰ fluorescence quantitative PCR method and GB method( GB4789.7-2013).The limit of PCR-ELISA method was close to SYBR GreenⅠfluorescence quantitative PCR method whose limit was 7.3×10 CFU/mL.While PCR-ELISA method wasmore economical and has a same accuracy.This study showed that PCR-ELISA method has the potential to be applied in rapid detection of Vibrio Parahemolyticus in seafood.