Development of molecular detection system of Cronobacter sakazakii based on the rpoB gene and its application to environment-monitoring

The present research introduces our work in developing a molecular detection system of Cronobacter Sakazakii based on the rpoB Gene and its application to the environment-monitoring practice.Cronobacter sakazakii is a human opportunistic pathogen which can cause life-threatening neonatal infections.It is foodborne with the primary source of infection from human inhabitation environment.Therefore,it is of great significance for scientists to detect and identify it.It is for this purpose that we are prompt to come to meet such an emergency and developed a Real-time PCR based system.The specific primers in the method were designed based on the non-conservative region of rpoB sequences by bioinformatics analysis.We have chosen two Cronobacter sakazakii strains and 12 closely related Enterobacter and Cronobacter strains to evaluate its specificity.The results of research show that Cronobacter sakazakii has a positive reaction to 154 bp amplification while all the other closely related strains just have negative reaction.To evaluate its sensitivity,we have adopted pure Cronobacter sakazakii DNA at the concentrations of 10 pg/μL,1 pg/μL,100 fg/μL,10 fg/μL and 1 fg/μL to determine the limitation of the detection.The results of our research indicate that the Real-time PCR method can at least detect at least 10 fg/μL of the DNA reliably.However,conventional PCR method can only be used to evaluate the sensitivity of the detectable DNA concentration at about 10 pg/μL.In addition,when we characterized the method in terms of its sensitivity and specificity,we also conducted further analysis by using this pair of primers to detect some environmental water samples we had collected from the fourteen important drinking water sources in Zhejiang.The results of our investigation of the sources prove that such kinds of pathogen were able to be detected only in six of the fourteen.And,now,we used again some conventional methods to identify the pathogen further,and,in turn,verified some samples from the four of the six sources to be positive,while other samples from the fourteen sources—proved to be negative.Thus,the results imply that the Real-time PCR system developed by us could be used for sensitive and specific detection of the bacterium in the environmental water conditions.Moreover,practical application results also provide basic information for controlling its likely outbreak in the future.