Application of polymerase chain reaction (PCR) and TaqMan (TM) PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces. has been suggested as a good indicator of animal (as opposed to human) faecal conta mination of aquatic environments. However, conventional detection methods l imit 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 faeca l samples is described. PCR and 5'-nuclease (TaqMan (TM)) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faeca l samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, pro ducing an amplicon of 443 bp which was specific for R. coprophilus. Sixty c ells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan (TM) PCR method. T he latter approach gave a linear quantitative response over at least four l og units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces bu t 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. (C) 2001 Publi shed by Elsevier Science B.V.