Isolation Of Bacteria And Es Tab.Lishment Of An EMA-qPCR Method To Detect Viable Cells Of Shewanella Putrefaciens In Frozen Shrimp

It has been reported that Shewanella putrefaciens is the spoilage bacteria in Penaeus Vannamei,and the Shewanella putrefaciens detection currently mainly relies on traditional plate culture method and ordinary PCR to detect.However,traditional plate culture method is cumbersome and time consuming and ordinary PCR cannot distinguish between dead bacteria and live bacteria.Combining EMA with traditional PCR or q PCR can selectively amplify live bacterial DNA and can quickly and accurately distinguish between dead bacteria and live bacteria.The method is able to effectively reduce false positives generated by traditional PCR or q PCR detection.In this study,an EMA-q PCR method was es Tab.lished for the rapid detection of live Shewanella putrefacien.The main results are as follows:1.Isolation and identification of bacteria in frozen shrimpA total of 400 frozen shrimps were collected from different regions in Chengdu and184 isolates were obtained from the shrimp.The 16S r DNA gene of these strains were sequenced and their sequencing were identified by NCBI BLAST.The results showed that the 184 isolates included 11 species.The proportion of Shewanella putrefaciens and Vibrio parahaemolyticus was the highest,and each of them was 48 strains.2.Es Tab.lishment of an EMA-q PCR method to detect viable cells of Shewanella putrefaciensThe specific primers and probe were designed based on the conserved gyr B gene.The pretreatment conditions including EMA of different concentrations and irradiating times were optimized.The detection limit to viable bacteria and test results in different mix proportions of bacteria of this method were confirmed.The detection specificity was evaluated by using 11 different bacteria.The results showed that the optimum operating condition is EMA with final concentration 20?g/m L and to incubate 20 min.The detection limit was 10 CFU per reaction and the maximum inhibitory concentration of dead bacteria was 1×10~5CFU/reaction.EMA-q PCR could completely inhibit the PCR amplification from DNA derived from dead cells,but no inhibition to viable cell in different mix proportions of bacteria.The results of specificity test showed that this method could effectively distinguish sebacillus putrescens from other bacteria with good specificity.The EMA-q PCR method es Tab.lished in this work can rapid and accuracy distinguish between dead and living bacteria.3.Application of EMA-q PCR in frozen shrimpA total of 202 frozen P.vannamei samples were collected from various aquatic markets and supermarkets in different districts and counties of Chengdu.Shewanella putrefaciens were identified by traditional plate method combination with 16S r DNA identification,ordinary q PCR and EMA-q PCR in frozen shrimp,respectively.The results showed that Shewanella putrefaciens were detected in 32,24 and 38 samples of shrimp by EMA-q PCR,plate culture and q PCR,respectively.EMA-q PCR can not only overcome the defects of traditional plate method,but also avoid the insufficiency of ordinary q PCR which can not distinguish live bacteria.It can be promoted in production and its conducive to the rapid diagnosis of spoilage shivatella...