Daniel L. Kacian

Chemiluminescent DNA probes: A comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays

Nucleic acid hybridization has the potential to markedly improve the diagnosis of infectious and genetic diseases. Recently, chemiluminescent hybridization assays using acridinium esters and stabilized dioxetanes have been described with sensitivities comparable to those obtained with radioactive labels. Acridinium esters are used as direct labels that are attached to the probe throughout the hybridization reaction. Methods have been developed for labeling DNA probes with acridinium esters at high specific activity and for stabilizing the label under the relatively harsh conditions of hybridization reactions. The label does not affect the kinetics of the hybridization reaction or the stability of the resulting hybrid. The label emits light upon exposure to alkaline peroxide; thus, the assay format can be an extremely simple one. The acridinium ester labels are stable in storage and exhibit extremely rapid light-off kinetics which permit reading large numbers of samples within a brief period as well as limiting the contribution of background signal. A special property of acridinium esters allows chemical destruction of the label when it is present on unhybridized probe, whereas the label is stable to this process when the probe is hybridized. This behavior forms the basis of techniques to minimize assay background signals and allows a homogeneous assay format which does not require physical separation of hybridized and unhybridized probe. The adamantyl-stabilized 1,2-dioxetanes have been used to produce high-sensitivity detection systems for clinical assays. The probe is labeled with enzymes such as alkaline phosphatase or beta-D-galactosidase that hydrolyze the dioxetane derivative to produce a chemiluminescent molecule. As with other enzyme-based labeling systems, the signal increases with time, allowing greater sensitivity to be achieved with longer incubations. The amount of light generated is sufficient to expose sensitive photographic film with extended incubation; therefore, convenient assay formats not requiring instrumentation can be used. Excellent analytical sensitivities have been reported, and by using labels with different light emissions and/or different enzymes on the probes, it is possible to distinguish multiple target sites within a single assay. Because the label is suited for use with solid supports such as polyacrylamide gels, membrane filters, or microscope slides, applications include DNA sequencing, dot and Southern blot hybridizations, and in situ hybridization.

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.