The Study Of Detection And Quantification Of Enterococci By Real-Time Quantification PCR

Objective: There are a lot of active and complex bacteria in human intestine. Though enterococci is not the most important bacteria that maintain the microecological balance of intestine.Ever growing evidences in vivo and in vitro have shown that administration of enterococci preparations and related products are able to reduce serum cholesterol, offering potential benefits to hypercholesterol individuals. However, the three different mechanisms by which enterococci induced the lowing of cholesterol have been established due to the diverse scientific reports from both at home and abroad. They are assimilation of cholesterol; deconjugation of bile salts;other mode of action like inhibiting the key enzymes required for biosynthesis of cholesterol. Some problems associated with research into the decrease of cholesterol by enterococci were also considered. Enterococci strain can regulate the level of cholesterol. Thus, rapid detection of enterococci in the feces sample is of importance judgement the microecological balance of intestine and also in assessing the quality of entercoccus curative effect. However, conventionnal methods for enterococcus detection based on cultivation often require 1 to 2 days. Additionally, growing bacteria in artificial media contributes to the poor culturability of injured and stressed organisms. Our purpose is to use realtime quantitative PCR found a new method that can quantify enterococci in feces accurately and quickly.Method: There are two parts in the experiment:One part is using TaqMan real time quantitative PCR assay .The probes and primers were designed and synthesized according to the conserved gene sequence of enterococci 23SrDNA available in GeneBank, and then reaction parameters were optimized to develop a real time TaqMan quantitative PCR assay.The samples were detected by using the established TaqMan real time quantitative PCR assay with DNA purifying . We also comparethe correlation between TaqMan real time quantitative PCR assay and traditional diluting quantification.The other part is using SYBRGreenI realtime quantitative PCR assay. The primers were designed and synthesized according to the conserved gene sequence of enterococci 23SrDNA available in GeneBank , and then reaction parameters were optimized to develop a SYBRGreenI realtime quantitative PCR assay. The samples were detected by using the established SYBRGreenI quantitative PCR assay with DNA purifying. We also compare the correlation between SYBRGreenI real time quantitative PCR assay and traditional diluting quantification.Results: The developed TaqMan real time quantitative PCR assay could detect 6 copies/reaction of plasmid DNA. The developed SYBRGreenI real time quantitative PCR assay could detect 7 copies/reaction of plasmid DNA and its while the results of the quantitative PCR were the same as those of the routine PCR.There is no obvious difference between realtime PCR quantification in feces and cultured bacteria quantification(P>0.05). There is no obvious difference between inflammatory diarrhea in feces and health adult. (P>0.05) There is no obvious difference between TaqMan real time quantitative PCR assay and SYBRGreenI real time quantitative PCR assay (P>0.05).Conclusions:Overall, the ability real time quantitative PCR to detect enterococci rapidly and quantitatively in the various feces samples represents a considerable advancement and a great potential for environmental applications.