Rapid and sensitive detection of MS2 coliphages in wastewater samples by quantitative reverse transcriptase PCR.
Coliphage MS2 is used in place of pathogens in many studies and is considered one of the indicators of pathogenetic viruses in wastewater. We developed a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay to quantify MS2 coliphages in treated wastewater samples. The format used was SYBR Green. The assay included an internal control to disclose the presence of PCR-product inhibitors. The method had a wide dynamic range (8 logs) with a correlation coefficient of 0.999 and is capable of detecting as few as 4x10(2) genome equivalents/100 ml of wastewater sample. The method was validated by using artificially contaminated water samples. The validated method was then applied to naturally contaminated samples collected in a wastewater treatment plant and the results were compared with those obtained by a plaque assay. In comparison with the plaque assay the PCR-method yielded viral counts about 1.5 orders of magnitude higher. The entire detection method, including sample processing and real-time PCR amplification, was completed within 4 hours, making it a rapid single-day method.
Gentilomi
Giovanna Angela
GA
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Ozzano Emilia, Italy. giovanna.gentilomi@unibo.it
Cricca
Monica
M
De Luca
Giovanna
G
Sacchetti
Rossella
R
Zanetti
Franca
F
D052160:Benzothiazoles; D003959:Diamines; D005618:Fresh Water / Q000821:virology*; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013997:Time Factors; D010948:Viral Plaque Assay
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I