Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase--polymerase chain reaction.
Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.
Tsai
Y L
YL
Environmental Sciences Laboratory, County Sanitation Districts of Orange County, Fountain Valley, CA 92708, USA. yltsai@uci.edu
Parker
S L
SL
D004783:Environmental Microbiology; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012623:Seawater / Q000821:virology*; D012722:Sewage / Q000821:virology*
D012722:Sewage