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OWML: Current Projects

Project Title: Evaluation of rapid viability polymerase chain reaction (RV-PCR) for Bacillus globigii spores in post-disinfected water concentrated by ultrafiltration

Project chief:  Rebecca Bushon

Project support:  Christopher Kephart, Erin Stelzer and Amie Brady

Cooperators:  U.S. Environmental Protection Agency and National Homeland Security Research Center

Project duration:  2012 – 2014

Introduction and problem:
Rapid detection of biological warfare agents in the event of a bioterrorist attack is necessary to quickly determine the risk to the public and facilitate the decontamination process. Following decontamination after release of a biological agent, rapid viability methods are needed to ensure successful cleanup and the absence of infectious agents. By use of molecular methods such as quantitative PCR (qPCR), results can be obtained in approximately 2 hours following sample filtration and concentration; however, results do not provide information on viability of organisms. Current cultural methods for determination of viability require days for confirmed results..

This study will test a rapid viability qPCR (RV-PCR) method for the detection of Bacillus globigii spores, as a surrogate for Bacillus anthracis. The method will be tested in post-disinfected, large-volume drinking water samples that are concentrated by ultrafiltration. Results from the rapid viability method will be compared to traditional culture methods. This study will provide information on the potential interference of the decontamination method with the RV-PCR method, and will evaluate methods of mitigating inhibition.

Goals and objectives:
The goal of the project is to test a rapid viability qPCR method for the detection of Bacillus globigii spores in large volume post-disinfected waters. Specific objectives are to 1) develop the capability to run RV-PCR at the OWML and test the method in small volume reagent-grade water samples, 2) test the method before and after chlorine disinfection at different contact concentrations and times, 3) test RV-PCR in post-disinfected large volume samples using the UF concentrator, and 4) address inhibition issues by evaluating cleanup steps, including IMS.

Objective 1: The OWML will receive hands-on training from the Lawrence Livermore National Laboratory (LLNL) on the RV-PCR method. Objective 2: Small volume samples spiked with Bacillus globigii spores will be analyzed by RV-PCR before and after chlorine disinfection. Various chlorine contact times will be evaluated. Objective 3: Large volumes of water will be spiked with Bacillus globigii spores, disinfected with chlorine, and then concentrated by ultrafiltration (UF). Varying amounts of water, ranging from 10 L to 100 L will be analyzed. Objective 4: If inhibition occurs following concentration by UF, methods to clean up samples, such as IMS, will be evaluated.

U.S. Environmental Protection Agency, 2011, Comparison of Ultrafiltration Techniques for Recovering Biothreat Agents in Water. EPA 600/R-11/103 October 2011. United States Environmental Protection Agency, Office of Research and Development, Washington, D.C.

U.S. Environmental Protection Agency, 2009, Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events (SAM). EPA/600/R-04/126E September 2009. Cincinnati, Ohio: United States Environmental Protection Agency, National Homeland Security Research Center.