Investigation of transcription-mediated amplification as a rapid test method for Enterococci in recreational water.

Growth-based methods for detecting faecal contamination of recreational water require at least 24 hours to yield results, which can delay detection, action and remediation, if required. Such delays can put human health at risk while closing beaches inappropriately can cause unnecessary economic losses. There is a requirement for more rapid methods to facilitate the management of pollution events. In this study, undertaken in our respective laboratories, a molecular assay based on transcription-mediated amplification (TMA) technology was compared with established growth-based methods. The assays were used to quantitate enterococcal faecal indicator bacteria (relative to the legislative threshold of 104 colony forming units/100 mL) in 138 water samples collected from 41 different locations, representative of different recreational water types routinely sampled in Southern California. The results demonstrate the efficacy of the TMA assay for the detection of Enterococcus species in recreational water as a rapid alternative to traditional methods.

Rapid tests for detection and quantitation of Enterococcus contamination in recreational waters.

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.