title: Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.
abstract: Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter(-1).
authors: Biology Department, Middle East Technical University, Ankara 06531, Turkey.
authors: Bilgin
authors: B
authors: Taskin
authors: Ayse Gul
authors: AG
authors: Gozen
authors: Metin
authors: M
authors: Duran
references: Appl Microbiol Biotechnol. 2010 Mar;86(1):377-84
references: 20024544
references: J Microbiol Methods. 2006 Nov;67(2):310-20
references: 16753236
references: J Microbiol Methods. 2008 Feb;72(2):180-4
references: 18160156
references: Water Res. 2009 Nov;43(19):4790-801
references: 19540546
references: Appl Environ Microbiol. 2007 Aug;73(16):5111-7
references: 17586667
references: J Appl Microbiol. 2008 Oct;105(4):1178-85
references: 18624747
references: Biotechniques. 2003 Apr;34(4):804-8, 810, 812-3
references: 12703305
references: J Microbiol Methods. 2008 Mar;72(3):275-82
references: 18280599
references: J Appl Microbiol. 1998 Jun;84(6):988-98
references: 9717283
references: Mol Cell Probes. 2004 Dec;18(6):437-41
references: 15488384
references: Appl Environ Microbiol. 2010 Jul;76(13):4318-26
references: 20472736
references: Appl Environ Microbiol. 2001 Jul;67(7):3122-6
references: 11425731
references: J Clin Microbiol. 2008 Jul;46(7):2305-13
references: 18448692
references: Int J Food Microbiol. 2003 Apr 25;82(2):143-51
references: 12568754
references: Appl Environ Microbiol. 2006 Mar;72(3):1997-2004
references: 16517648
references: Appl Environ Microbiol. 2007 Dec;73(24):8028-31
references: 17933922
references: Res Microbiol. 2007 Jun;158(5):405-12
references: 17449228
references: Water Environ Res. 2006 Sep;78(9):1005-12
references: 17120460
references: Microbes Infect. 2000 Oct;2(12):1523-35
references: 11099939
references: Methods Mol Biol. 2004;263:33-44
references: 14976359
references: Water Res. 2007 Feb;41(3):571-80
references: 17188734
references: Appl Environ Microbiol. 2000 Oct;66(10):4266-71
references: 11010869
references: Water Res. 2006 Sep;40(16):3037-3044
references: 16908046
references: Appl Environ Microbiol. 1991 Apr;57(4):1013-7
references: 2059028
references: Appl Environ Microbiol. 2008 Apr;74(8):2537-9
references: 18296534
references: Curr Microbiol. 2011 Feb;62(2):374-81
references: 20680282
references: J Virol Methods. 2010 Sep;168(1-2):228-32
references: 20599560
references: Appl Environ Microbiol. 2009 May;75(9):2936-9
references: 19270144
references: Water Res. 2007 Feb;41(3):665-73
references: 17107701
references: Appl Environ Microbiol. 2009 May;75(9):2940-4
references: 19270114
references: Appl Environ Microbiol. 2005 Feb;71(2):1018-24
references: 15691961
references: J Orthop Res. 2009 Sep;27(9):1243-7
references: 19322790