title: Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.
abstract: Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.
authors.affiliation: Christchurch Science Centre, Institute of Environmental Science and Research (ESR) Ltd., PO Box 29-181, Christchurch, New Zealand. marion.savill@esr.cri.nz
authors.forename: M G
authors.initials: MG
authors.lastname: Savill
authors.forename: S R
authors.initials: SR
authors.lastname: Murray
authors.forename: P
authors.initials: P
authors.lastname: Scholes
authors.forename: E W
authors.initials: EW
authors.lastname: Maas
authors.forename: R E
authors.initials: RE
authors.lastname: McCormick
authors.forename: E B
authors.initials: EB
authors.lastname: Moore
authors.forename: B J
authors.initials: BJ
authors.lastname: Gilpin
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000032:analysis; D003670:Deer; D004372:Ducks; D005243:Feces / Q000382:microbiology*; D006736:Horses; D006801:Humans; D009893:Opossums; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D012240:Rhodococcus / Q000145:classification / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012756:Sheep; D013552:Swine; D019914:Taq Polymerase
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase