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Application of real-time quantitative PCR for the detection of selected bacterial pathogens

Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

Application of real-time quantitative PCR for the detection

of selected bacterial pathogens during

municipal wastewater treatment

K.E.Shannon a ,D.-Y .Lee b ,J.T.Trevors a,⁎,L.A.Beaudette c,⁎

a Department of Environmental Biology,University of Guelph,Guelph,ON,Canada N1G 2W1

b Aquati

c Ecosystem Management Research Division,Science an

d Technology Branch,Environment Canada,867Lakeshor

e Rd.,

Burlington,ON,Canada L7R 4A6

c

Environmental Technology Centre,Science and Technology Branch,Environment Canada,335River Road,Ottawa,ON,Canada K1A 0H3Received 26October 2006;received in revised form 15February 2007;accepted 25February 2007

Available online 25April 2007

Abstract

Bacteria were detected at five stages of municipal wastewater treatment using TaqMan®real-time quantitative PCR (qPCR).Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater,including Aeromonas hydrophila ,Bacillus cereus ,Clostridium perfringens ,Enterococcus faecalis ,Escherichia coli ,E.coli O157:H7,Helicobacter pylori ,Klebsiella pneumoniae ,Legionella pneumophila ,Listeria monocytogenes ,Pseudomonas aeruginosa ,Salmonella sp.,and Staphylococcus aureus .The sensitivity of the assay was 100fg of genomic DNA (=22gene copies),based on a standard curve generated using A.hydrophila purified DNA.Samples from five stages of wastewater treatment were collected,including raw wastewater,primary effluents,mixed liquor,waste activated sludge and final effluents.In duplicate samples, E.coli ,K.pneumoniae ,C.perfringens and E.faecalis were detected throughout the wastewater process,and their numbers decreased by

3.52–3.98,

4.23–4.33,3.15–3.39,and 3.24orders of magnitude respectively,between the raw wastewater and final effluent stage.This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines.

©2007Elsevier B.V .All rights reserved.

Keywords:Detection;Disease;Enumeration;Environment;Microorganisms;Pathogens;Public health;Real-time quantitative PCR;Wastewater;Water

1.Introduction

Waterborne diseases are a major world-wide threat to

public health,despite significant advances in water and

wastewater treatment technology.Waterborne disease is

estimated to be responsible for 4.0%of all deaths and 5.7%

of the total disease burden worldwide (Prüss et al.,2002).

Though many of these infections occur in developing

countries with lower levels of sanitation and less

Application of real-time quantitative PCR for the detection of selected bacterial pathogens

public Science of the Total Environment 382(2007)121–

Application of real-time quantitative PCR for the detection of selected bacterial pathogens

Application of real-time quantitative PCR for the detection of selected bacterial pathogens

http://doc.xuehai.net/locate/scitotenv

⁎Corresponding authors.Trevors is to be contacted at Tel.:+15198244120x53767;fax:+15198370442.Beaudette,Tel.:+16139491336;fax:+16139900173.E-mail addresses:jtrevors@uoguelph.ca (J.T.Trevors),

Lee.Beaudette@ec.gc.ca (L.A.Beaudette).

0048-9697/$-see front matter ©2007Elsevier B.V .All rights reserved.

doi:10.1016/j.scitotenv.2007.02.039

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