Rapid Evaluation Of Water Disinfection Efficacy Based On Reverse Transcription-PCR Combined With MRNA

ObjectiveTo develop an mRNA-based reverse transcription polymerase chain reaction (mRNA-RT-PCR) assay for the specific detection of viable Escherichia coli, and to observe the effect of the RT-PCR method in rapid detecting viable E.coli of water disinfection test in laboratory.Methods1. Escherichia coli8099was selected and serially diluted10-fold for this study; the expression of three genes, tuf A, lac Z and uid A, was examined to determine a suitable target for amplification. The reaction and conditions were improved, and then a PCR was carried out with40cycles. The products were electrophoresed on a2%(wt/vol) agarose gel.2. According to suspension quantitative germicidal test in Technical Standard For Disinfection (2002Edition), sodium dichloroisoc-yanurate(SDIC) and chlorine dioxide commonly used in water disinfection were chosen in this study. The viable E.coli, DNA and RNA were detected before and after disinfection with the traditional culture method, the common PCR and the RT-PCR method.3. The sample was exposed to low concentration of disinfectant, then incubated at4℃for12hours before examination by RT-PCR. The mRNA signals were detected at five different points of time within12hours for the effectiveness test of the new method applied in the evaluation of water disinfection efficacy.Results1. The target gene of uid A was obtained, and the sensitivity of RT-PCR detection was3.6×103cfu/mL for viable E.coli. Overall, the assay could be completed in6～8hours, and had the advantages of good specificity and homology.2. Exposed to SDIC (40mg/L) for5mins, the E.coli was killed by the traditional culture method, negative by the new RT-PCR method, while strong positive by the common PCR. Additionally, when exposed to SDIC (20mg/L) for5mins, concentration of the suspension of E.coli was4.0×103cfu/mL by the culture method, weakly positive result by the RT-PCR method which signified low amounts of live E.coli, while strong positive result by the common PCR which signified high amounts of E.coli DNA.3. Exposed to chlorine dioxide (4mg/L) for5mins, the E.coli was killed by the traditional culture method, negative by the new RT-PCR method, while strong positive by the common PCR. Additionally, when exposed to chlorine dioxide (0.8mg/L) for5mins, concentration of the suspension of E.coli was8.9X103cfu/mL by the culture method, weakly positive result by the RT-PCR method which signified low amounts of live E.coli, while strong positive result by the common PCR which signified high amounts of E.coli DNA.4. The E.coli mRNA signal persisted for12h at4℃after exposure to low concentration of disinfectant, the concentration of the suspension did not change within12hours, and the brightness of the mRNA signals always kept at the same level correspondingly.Conclusion1. In the laboratory conditions, the new RT-PCR method can detect3.6×10cfu/mL viable E.coli rapidly and accurately.2. When the remaining number of live E.coli is above the detection limit of the new method, the RT-PCR method can be qualitatively used in the evaluation test of water disinfection effect in laboratory. On the contrary, the common PCR cannot be.3. The mRNA of E.coli degrades quickly after exposure to SDIC or chlorine dioxide. The results of the RT-PCR are correct immediately after water disinfection.