Development Of A Real-time QPCR Method (TaqMan Probe Based) For Detection And Enumeration Of Escherichia Coli In Water

Objectives：1． To develop a Real-Time qPCR Method (TaqMan probe) for Detection andEnumeration of Escherichia coli．2． To find a optimized combinatorial method, which was sensitive, accurate and rapidfor Escherichia coli concentration and DNA extraction, so that we can applyReal-Time qPCR method in Water.3． To comfirm the accuracy of Real-Time qPCR Method, the result both of PCR andof conventional culture for natural water samples were compared and statisticallyanalyzed.Methods：1． Specific primers and probe were designed according to targeting the ydiJ gene of Escherichiacoli. The primer taxonomic specificity and sensitivity were confirmed by polymerase chainreaction (PCR).2． A Plasmid DNA Calibration standards containing a segment of the ydiJ gene werecreated by linking the PCR products and the T-vecter．3． The reaction systems and conditions for PCR were optimized. Then Standardcurves were created and evaluated.4． A combinatorial method was optimized for Escherichia coli concentration and DNA extractionby analyseing different methods.5．20natural water samples were detected by Real-Time qPCR method. The accuracy ofReal-Time qPCR Method was confirmed by comparing and statistically analyzing theresult both of PCR and of conventional culture.Results: 1. In this study, the primers showed good specificity with all the strains tested. All theEscherichia coli and Shigga were all positive, and9Non-E. coli strains were allnegative.2. The Plasmid DNA Calibration standards were purified and sequenced and comparedthe sequences by BLAST．the result showed that the Identities was90/91(99%),Gaps was0/91(0%)。，and the purity (OD260/OD280) of Plasmid DNA was1.81.3. Membrane filtration and beads extraction methods were used for Escherichia coliconcentration and DNA extraction.The linearity of quantitative PCR was linearfrom2.3×100～2.3×106CFU/mL (R2:0.996). The detection limit was estimated to be2.3CFU/mL.4. The results showed that the numbers obtained by RT-qPCR correlated with thosemeasured by MPN method, r=0.991, P<0.01.Conclusions:1. The RT-qPCR method is rapid and accurate technique with a good specificity forenumerating Escherichia coli in water samples．2. The RT-qPCR method is more suitable for enumerating Escherichia coli in watersamples contaminated with a middle and high level of fecal bacteria.3. The RT-qPCR method can be as a supplementary of the current conventional culturemethods for accurately monitoring and quikly assessing the level of fecal bacteria inwater samples.