The Study On The Typing Of Staphylococcus Aureus And Salmonella Based On MLMA

Food safety issues are directly related to environmental safety and human health.With the influence of accellerating globalization and environmental factors,food safety has become one of the most important public health problems in the world.Foodborne pathogens are one of the most important causes of food safety.Among the common foodborne pathogens which cause food safety in China,Staphylococcus aureus and Salmonella are two important foodborne pathogens.Staphylococcus aureus is widely distributed in the environment,and most Staphylococcus aureus strains produce Staphylococcal Enterotoxins(SEs).SEs are the main pathogenic factor which cause Staphylococcus aureus food poisoning and cause acute gastroenteritis.The type of SE is numerous and can be divided into about 20 types based on the different antigenicity.Therefore,it is very important to identify whether Staphylococcus aureus contains SE and the specific enterotoxin type for the diagnosis of food poisoning caused by Staphylococcus aureus.Currently,the cost of enzymelinked immunosorbent assay(ELISA)for the diagnosis of SE is high and SEA to SEE only can be identified.The common PCR method is time-consuming,easily contaminated,and false positives producing.So multiplex ligation reaction based on probe melting curve analysis(MLMA)for the identification of 16 Staphylococcus aureus enterotoxins was developed.As the results,the limit of MLMA ranged from 0.80ng/u L to 2.15ng/u L.The target genes were 100% specificity with no crossfluorescence signal,and the coefficient of variation(CV)was lower than 1%.Compared with the commoon PCR method,the sensitivity and specificity of MLMA was 95.4% and 100%,Kappa was 0.88.The MLMA for the identification of 16 Staphylococcus enterotoxins is rapid,accurate and specific.Salmonella is another important foodborne pathogen.Salmonella infection is among the top two foodborne diseases in the world and has become one of important food safety issues.Salmonella has a wide variety of serotypes,and there are more than 100 Salmonella serotypes closely related to human diseases.DifferentSalmonella serotypes have different genetic structures and different host types,resulting in the different epidemiological prevalence of Salmonella infectoins.Therefore,the rapid and accurate identification of Salmonella serotypes is of great significant for the prevention and control of Salmonella infection,including the risk factor analysis,clinical diagnosis and treatment of salmonellosis.The traditional Salmonella serotyping method is time consuming and the serum slide agglutination results are greatly guided by the subjective views,as the different operator’s observing the serum slide agglutination results is different.Multiplex PCR is easily contaminated and less serotypes are identified by multiplex fluorescent PCR.Furthermorethe,liquidphase chip technology is monopolized and expensive.In this study,the common 30 Salmonella serotypes were identified by MLMA.The limit of MLMA ranged from 1.04ng/u L to 1.56ng/u L.No cross reaction was detected by identifying the target 30 Salmonella serotypes,the other 34 Salmonella serotypes,one Escherichia coli,one Shigella and one S.mirabilis strain.The 431 Salmonella strains were analyzed to evaluate the specificity of the new developed assay with the comparison of the traditional serum slide agglutination assay.The results showed that the sensitivity and specificity of MLMA was 99.5% and 100%,the Kappa was 0.89,and the coefficient of variation(CV)was less than 1%.The system has achieved the purpose of quickly and accurately identifying the common 30 Salmonella serotypes and is expected to replace the traditional serological identification method to reduce workload and improve the accuracy.In summary,the multiplex ligation reaction based on probe melting curve analysis for the identification of 16 Staphylococcus enterotoxins and 30 Salmonella serotypes was developed.The assay is rapid,accurate and specific.It has a good application prospect for the rapid identification of two foodborne pathogens in food and clinical samples.