BackgroundThere is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.ResultsLoop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.ConclusionsLAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

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.

An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growthbased methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods.Enterococcus is the recommended fecal indicator bacterium for monitoring the presence of fecal contamination in recreational waters (5). Standards for the maximum acceptable level of Enterococcus bacteria in recreational waters have been established by the U.S. EPA (U.S. Environmental Protection Agency). The most common single-sample standard used for managing recreational waters is 104 most probable number (MPN) or CFU per 100 ml. Presently, most regulatory agencies worldwide use traditional growth-based tests, such as membrane filtration and multiple-tube fermentation, for the detection and quantitation of fecal indicator bacteria in water samples. These tests typically require 18 to 24 h to yield results.This report describes a rapid test for the quantitation of Enterococcus rRNA that yields a result within 4 h. The method involves filtration of the water sample, lysis of the target organism, and purification of rRNA using magnetic-particle target capture, followed by amplification of the purified rRNA using transcription-mediated amplification (TMA) technology. Homogeneous, real-time detection of amplicons is achieved by using a fluorescent oligonucleotide probe.TMA technology is both isothermal and rapid. This assay allows highly sensitive and specific quantitation using a standard curve to derive the equivalent of CFU-per-100-ml values. The assay does not detect nonenterococcal organisms that are known to cause false-positive results in some of the U.S. EPAapproved growth-based methods (2) or Enterococcus species that are underrepresented in human feces (3).The optimization and development of the TMA method for the detection of Enterococcus deliberately spiked into seawater have been described previously (3), and the present study expands on this research to apply the method to a wide range of ambient environmental water samples. The goal of this study was to compare the results obtained with the TMA assay to results obtained with two of the most commonly used reference methods for quantitation of Enterococcus bacteria in water samples, Enterolert (Idexx Laboratories, Inc