Application Of Ethidium Bromide Monoazide (EMA) And PCR Method To Detect Of Viable Salmonella In Chicken Meat

Ethidium bromide monoazide (EMA) is a kind of fluorescent dye to nucleic acid, and it has a specificity of covalently bind to extracellular DNA and DNA contained in dead or membrane compromised bacterial cells and inhibit those DNA from PCR amplification, but it doesn’t impact the DNA amplification of live bacterial ones. So PCR method combines with EMA could make the number of viable cells more accurately, and this method could get rid of the disadvantages of direct PCR method which cannot distinguish dead bacterial from live ones, and avoid false positive results. PCR technique combines with EMA has been used for the detection of variety of pathogenic bacteria, many research showed that the addition of EMA could not affect the DNA amplification of Vibrio parahaemolyticus, bowel Campylobacter, Zygosaccharomyces, Serratia mnarcescens, et al, however, this method could lead the loss of DNA from Escherichia coli O 157:H7, Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Streptococcus sobrinus, et al. Nonetheless, whether the EMA can restrain the DNA amplification of live Salmonella enteritidis and Salmonella typhimurium there was still no report. So in this study, EMA was used for the Salmonella enteritidis and Salmonella typhimurium detection, and we hope we can establish a method to detect the liv ebacteria only. The main contents and results are as follows:1. The establishment of the method to detect Salmonella enteritidis by EMA PCRIn this part, we first studied whether DNA amplification of Salmonella enteritidis would be effected or not by EMA, and then EMA was used in live/dead sample’s detection for EMA PCR method’s establishment, we found that the amplification of DNA extracted from the concentration of 2.75×107 CFU/mL of dead Salmonella enteritidis was totally restrained with 50 μg/mL EMA and exposure time of 10 min, which did’nt affect the sensitivity of Salmonella enteritidis detection. With the parameters selected in this method, the false positive results could be avoided.2. The establishment of the method to detect Salmonella typhimurium and Salmonella enteritidis in chicken breast by EMA RT-PCRIn this part,we used EMA combined with RT-PCR as method to detect salmonella in chicken meat.The results showed that live bacteria samples added with EMA to a final density of 50μg/mL, its Ct values had no significant difference from the untreated one (P>0.05). Whereas, with EMA treatment, Ct value of dead bacteria is significantly different from that of live bacteria(P<0.05) while had no obvious difference(P>0.05)from negative control one Thoes results demonstrated DNA amplification of Salmonella typhimurium and Salmonella enteritidis didn’t be influenced by EMA. When using EMA RT-PCR method which was established by specific primer of Salmonella typhimurium to detecte Salmonella typhimurium in chicken, we found the result comes closer to plate counting(P>0.05) than direct RT-PCR. Correspondingly, we got the same result when using EMA RT-PCR which was established by specific primer of Salmonella enteritidis method to detect Salmonella enteritidis in in chicken.3. The establishment of the method to detect mixed Salmonella in chicken breast by EMA RT-PCR Multiple PCR techniqueIn this part, we used three EMA RT-PCR methods which was established by universal primer of Salmonella, specific primer of Salmonella typhimurium and specific primer of Salmonella enteritidis to detect Salmonella in chicken meat. The results were as follows: when just added one primer, the EMA RT-PCR method which was established by universal primer of Salmonella can make the detection of the number of live salmonella more accurately,and its results had no significant difference with plate counting ones (P> 0.05);and the results of using EMA RT-PCR method which was established by specific primer of Salmonella typhimurium and specific primer of Salmonella enteritidis were also had no significant difference with plate counting ones(P> 0.05). But when detected live/dead samples using three primers, the results had significant difference between EMA RT-PCR methods and plate counting (P< 0.05). So, in order to improve the accuracy of the method we can optimize the parameters such as reaction system or reaction factors.