title: Rapid tests for detection and quantitation of Enterococcus contamination in recreational waters.
abstract: Presently, growth-based tests are used for the detection and quantitation of microbiological contaminants in the environment. These tests take a minimum of 24 h to generate a result, which compromises the ability to take the most appropriate action. This report describes a rapid test for Enterococcus in recreational water as an indicator of faecal contamination. This method involves (1) isolation and lysis of the target organism, (2) purification of ribosomal RNA (rRNA) from the lysate and (3) amplification and detection of the purified rRNA. rRNA is used as the target since, in contrast to DNA, there are hundreds to thousands of copies in the cell. The rRNA is purified from the lysate by target capture onto magnetic microspheres, which removes interfering substances present in the sample. The rRNA is then quantitated using transcription-mediated amplification (TMA) with real-time homogeneous detection of amplicon using a fluorescent oligonucleotide probe. Compared to polymerase chain reaction (PCR) amplification, TMA is isothermal, more rapid, and ideally suited to RNA detection. The test described here demonstrates sensitive detection and quantitation of enterococci over a wide dynamic range with a high level of analytical specificity. The latter is particularly important for accurate and relevant monitoring both for protecting public health and for source tracking. Many conventional microbiological tests are time-consuming, exhibit limited dynamic range and are known to lack specificity. This assay demonstrates the advantages achievable by the application of TMA of rRNA targets to current environmental testing challenges.
authors.affiliation: MLT Research Ltd, 5 Chiltern Close, Cardiff, UK, CF14 5DL.
authors.forename: Rhian
authors.initials: R
authors.lastname: Morgan
authors.forename: Ceri
authors.initials: C
authors.lastname: Morris
authors.forename: Kristin
authors.initials: K
authors.lastname: Livzey
authors.forename: James
authors.initials: J
authors.lastname: Hogan
authors.forename: Neil
authors.initials: N
authors.lastname: Buttigieg
authors.forename: Reinhold
authors.initials: R
authors.lastname: Pollner
authors.forename: Daniel
authors.initials: D
authors.lastname: Kacian
authors.forename: Ian
authors.initials: I
authors.lastname: Weeks
mesh_terms: D002457:Cell Extracts / Q000032:analysis; D016983:Enterococcus / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D015345:Oligonucleotide Probes / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012335:RNA, Ribosomal / Q000378:metabolism; D011998:Recreation; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014871:Water Microbiology; D014881:Water Supply / Q000032:analysis*
chemical_list: D002457:Cell Extracts; D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal