Q-249. Increasing the Precision and Accuracy of a Nucleic Acid Sequence Based Amplification (NASBA) Method for Detection and Enumeration of Enterococci from Coastal Waters Utilizing an Internal Control (IC) RNA Molecule

L. Poorvin1, S. S. Patterson1, M. L. Eldridge1, G. S. Sayler1, J. H. Paul2;
1Univ. of Tennessee, Knoxville, TN, 2Univ. of South Florida, St. Petersburg, FL.

The coastal ocean is an important economic and recreational resource that is constantly influenced by human activities. In 2006, there were more than 25,000 days of beach closings in the US due to fecal contamination. This was an increase of 28% over the previous year and the highest on record. Currently, health authorities monitor fecal contamination of recreational beaches via enumeration of membrane filtered coliform and enterococci bacteria. There is a strong correlation between exposure to elevated concentrations of enterococci in marine waters and increased prevalence of gastrointestinal diseases and other illnesses, making enterococci the indicator organism of choice for coastal monitoring. The main disadvantage with membrane filtration techniques is the long lag time, 24-48 hours, between sample collection and availability of results. To overcome this, we have previously developed a real-time nucleic acid sequence based amplification (NASBA) assay using primers and probe for the large subunit ribosomal RNA gene of enterococci which is conserved in Enterococcus species. To increase the accuracy and precision of this assay, we have designed and synthesized an internal control (IC) RNA molecule that shares the priming sites of the target sequence but contains a modified beacon site which is recognized by a second probe. With this IC-NASBA assay we are able to detect one enterococci colony forming unit (CFU) from experimentally spiked (100 ml) sea water samples; achieve a negative linear correlation (R2 = 0.948) between CFUs obtained via membrane filtration and time to positivity readings obtained with the NASBA assay; provide quantitative estimates of enterococci over at least five orders of magnitude; and amplify all positive samples within forty minutes. This IC-NASBA method also provides several advantages over PCR based fecal monitoring techniques. Because NASBA is isothermal (41°C), we have been able to link the assay to an inexpensive, field based handheld detector which will provide the ability for real-time sample analysis at the site in less than one hour. With these advantages NASBA can be a simple, quick and convenient alternative to conventional water testing methodologies.