Study On The Detection Of Live Vibrio Parahaemolyticus In Seafood By PMA-qSRCA

Vibrio parahemolyticus(Vp),an important food-borne pathogen,mainly exists in seafood products such as shrimp,crab,oyster,clam,scallop and marine fish.The conventional culture methods(GB 4789.7-2013)are time-consuming,laborious,and cannot detect viable but non-cultivable(VBNC)Vibrio parahaemolyticus.In spite of the considerable advantages of test preiod,sensitivity and special,molecular detection technology cannot remove the amplication singal of DNA from dead cells.PMA can prevent the amplification reaction by specificly penetrating damaged cell membranes of dead cells and embeding in the DNA molecules,thus eliminating the amplification signal interference of DNA from dead cells and improving the accuracy of detection of the molecular detection methods.Two primers were designed and screened to identify specific target sites on toxR gene of Vibrio parahaemolyticus.Real-time fluorescence saltatory rolling circle amplification reaction system(RF-SRCA)was established by adding EvaGreen fluorescent dye into SRCA reaction system,and the RF-SRCA reaction condition and reaction system was optimized.We combined RF-SRCA with PMA dyeand optimized the processing conditions of PMA dye to establish a PMA-qSRCA method for live bacteria detection.The specificity,sensitivity,limit of detection,limit of quantification and accuracy of the method were evaluated.Finally,this method was used for detection of actual samples.The optimized results of PMA-qSRCA detection method for live bacteria showed that:the appropriate reaction temperature was 62?,and the appropriate reaction system was as follows:0.50 mM for dNTPs,3.00 mM for Mg2+,1 × ThermoPol Reaction Buffer,0.50?M for FWP,0.50 ?M for RVP,3.56 ng/?L for DNA template,0.60 U/?L for Bst DNA polymerase,1 x EvaGreen and sterile deionized water to 20.00 ?L.The suitable treatment conditions of cells using PMA were as follows:all cells were treated with PMA at the concentration of 40 ?M for 8 min in the dark,and then illuminated for 15 min.The results of the primer specificity test showed that 12 strains of Vibrio parahaemolyticus presented positive results,while negative results were obtained from 29 non-Vibrio parahaemolytic strains.Therefore,the detection method has good specificity.The sensitivity of the PMA-qSRCA method was evaluated by using different concentrations of pure bacteria.Oyster samples with different concentrations of Vibrio parahaemolyticus were used to evaluate the detection limit and quantitative limit of the PMA-qSRCA method,and comparisons with the PMA-qLAMP and PMA-qPCR methods were carried out.The results showed that the sensitivity of PMA-qSRCA method was 3.20× 100 CFU/mL,which was 10-fold higher than PMA-qLAMP method and 100-fold higher in comparison with PMA-qPCR method.The detection limit of PMA-qSRCA method was 2.08 ? 101 CFU/g,which was 10-foldlower than PMA-qLAMP method and 100-fold lower compared with PMA-qPCR method.The lower limit of quantification of PMA-qSRCA method was 2.08 × 102 CFU/g,which was 10-fold lower than PMA-qLAMP and PMA-qPCR.The accuracy of PMA-qSRCA detection method of live bacteria was evaluated by comparing the detection effectiveness of PMA-qSRCA method with that of plate culture method and qSRCA method.The results showed that,there was no significant difference(p>0.05)in viable bacteria mixtures with different proportions between the plate count and PMA-qSRCA methods.The results of qSRCA method and the other two methods were significantly different(p<0.05)at the viable bacteria concentrations ranging from 0%to 80%.Whereas there was no differences between the three methods(p>0.05)when the cell mixture was all viable.Therefore,the PMA-qSRCA method has high accuracy and can be employed in the detection of viable Vibrio parahaemolyticus.In summary,the PMA-qSRCA method established in this experiment has significant advantages characterized by specific,sensitive,accurate and fast,which is suitable for the detection of vibrio parahaemolyticus.Moreover,it provides a new method and has great application potential for rapid detection and screening of live foodborne pathogens.