Rapid Detection Of Salmonella In Chicken Meat Without Enrichment By Rti-LAMP

Salmonella is one of the important pathogen in enterobacteriaceae,people can be infected through eating contaminated vegetables or under-cooked meat food,then causing foodborne disease,which include vomiting,fever,diarrhea and other symptoms.with the food safety issues caused by the pathogenic microorganisms highlighted,as an important technical support to guarantee for food safety,the detecting technology,especially rapid detecting technology for pathogenic bacteria play an increasingly important role in food production,circulation process control and supervision as well as import and export trade.Therefore,the research and development of accurate,sensitive,simple,and rapid detection methods for salmonella and other pathogenic microorganisms is extremely important.Firstly,this study was conducted to investigate the pollution rate of chicken in five regions in Nanchang.The result as follow,75 chicken samples were detected by salmonella culture,after enrichment,selective enrichment and biochemical experiments,further application of molecular biology identification.A total of 3 salmonella positive chicken samples were detected,the salmonella positive rate were 4%,all the experiments can be completed in 4 days.Secondly,in this study,a real-time loop amplified DNA assay system(Rti-LAMP)was developed for the rapid detection of Salmonella enterica on the chicken.It showed that the lowest level of 6 CFU/reaction for pure Salmonella culture could be detected and finished in 45 minutes by the Rti-LAMP targeting the inv A gene using Midori Green as nucleic acid dye.Furthermore,seeding 25 g portions of chicken with various numbers of Salmonella CFU,followed enrichment culture at 37? C for 4 h,then filtration through Whirl-Pak bag and pelleting the bacterial cells by centrifugation at 13,000 g.The resulting pellets were suspended in saline and processed for cell lysis and DNA purification.2 ?L purified DNA samples was incorporated into 25 ?L Rti-LAMP reactions at 65 ? for 60 min.The lowest level 450 CFU/g of Salmonella consistently detected by the Rti-LAMP assay.The entire assay could be completed in 7 h.21 chicken samples purchased from market were detected by the Rti-LAMP assay as the control of Salmonella culture.The same one sample was Salmonella positive by the both of assays.It suggested that the sensitivity of the Rti-LAMP assay was as well as that of Salmonella culture on detection of chicken sample,but the former need only 7 h and was much more rapid than that of the latter?Lastly,in order to further shorten the detection time and investigate the effect of non-pre-enrichment on the detection of Salmonella in chicken,and to study the effect of Rti-LAMP on the sensitivity of Salmonella in chicken by using the bentonite coated activated carbon to adsorb the chicken sample to remove the soluble DNA polymerase inhibitor in chicken.The results showed that: the activated carbon was coated withbentonite with the mass ratio of 10%,then 4 g of treated activated carbon were used to absorb 25 g of chicken and the bacterial recovery rate was 98%.The absorbance(OD600)of the sample extract decreased from 0.91 to 0.52,indicating that the activated carbon did not adsorb the bacteria but could remove the soluble components of the chicken.The bacterial DNA was prepared by recovering the purified bacteria and detected by Rti-LAMP.The detection sensitivity of Salmonella in chicken was 23 CFU / g,which was nearly 20 times higher than that of the activated carbon(Tc),but the whole detection process was reduced from 7 h to about 4 h.A standard curve was generated by plotting the Tp values(min)against the log of S.enterica seeded onto chicken meat.Thirty-four chicken samples purchased from market were detected by both the Rti-LAMP assay and Salmonella culture.Six samples were positive of Salmonella detected by Rti-LAMP assay which of only one sample positive by the culture.It suggested that the sensitivity of the Rti-LAMP assay was better than that of the culture,furthermore the former could be completed in 4 h that was much more rapid than that of the latter.