Duplex Real-Time PCR Method For The Differentiation Of Cronobacter Sakazakii And Cronobacter Malonaticus

Cronobacter spp.are an important Gram-negative opportunistic food-borne pathogens that cause serious human illness,especially in the newborn.The mortality rates of infections can vary between 40% and 80%.The current taxonomy of the Cronobacter spp.group contains seven closely related species: Cronobacter sakazakii,Cronobacter muytjensii,Cronobacter turicensis,Cronobacter malonaticus,Cronobacter dublinensis,Cronobacter condiment and Cronobacter universalis.C.sakazakii and C.malonaticus are more important than other species of Cronobacter,as they are the most isolated strains,according to some reports.Until now,the isolated Cronobacter strains recorded in the MLST database(http://pubmlst.org/cronobacter)indicated that the isolated percentage of C.sakazakii was 65.5% and of C.malonaticus was 11.99%.So it is important and necessary to identify the two species.We developed a real-time PCR method for identifying C.sakazakii and C.malonaticus from the genus Cronobacter.The results suggested that the developed real-time PCR was more rapid and more sensitive for the identification of C.sakazakii and C.malonaticus compared with traditional method.In our study,we first identified whether the 112 strains used in our study were all Cronobacter spp.using the MMS probe and primers according to a fluorescence quantitative PCR test.We confirmed the 112 strains used in our study were all Cronobacter spp.,then we identified C.sakazakii and C.malonaticus from the 112 strains using the duplex real-time PCR method developed in our study.In our study,two pairs of primers and probes were designed targeting 16 S rRNA and fusA gene,respectively.The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter spp.,including 56 C.sakazakii,32 C.malonaticus,16 C.dublinensis,6 C.turicensis,and 2 C.muytjensii.The results showed that C.sakazakii and C.malonaticus were all correctly identified,consistent with the results of another method by analyzing the clustering of the fusA sequence.Then we detected the sensitivity of the system using the pure culture and artificially contaminated PIF of C.sakazakii and C.malonaticus.The results showed,the detection limit for pure culture was 102 CFU/ml and 103 CFU/g for artificially contaminated rehydrated powdered infant formula.We also constructed the plasmid standard and drawn the standard curve for absolute quantitative of the detection results.The gradient concentration standard plasmid of real-time PCR results showed that there had a good liner relationship between the cycle threshold(Ct)and the logarithmic amount of initial template.The standard curve showed that the CSM-CTD detection limit for pure culture of ATCC 29544 was 131 copies/ul,the CS-CMA detection limit for pure culture of ATCC 29544 was 16 copies/ul and the CSM-CTD detection limit for pure culture of DSM 18702 was 136 copies/ul.Therefore,the developed real-time PCR was a rapid,sensitive,and reliable method for the identification of C.sakazakii and C.malonaticus.The method can also be used for infant formula in the rapid screening and identification of C.sakazakii and has great application value and popularization value.