title: Rapid and sensitive detection of MS2 coliphages in wastewater samples by quantitative reverse transcriptase PCR.
abstract: 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.
authors.affiliation: Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Ozzano Emilia, Italy. giovanna.gentilomi@unibo.it
authors.forename: Giovanna Angela
authors.initials: GA
authors.lastname: Gentilomi
authors.forename: Monica
authors.initials: M
authors.lastname: Cricca
authors.forename: Giovanna
authors.initials: G
authors.lastname: De Luca
authors.forename: Rossella
authors.initials: R
authors.lastname: Sacchetti
authors.forename: Franca
authors.initials: F
authors.lastname: Zanetti
mesh_terms: 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
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I