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Optimization of a Rapid Viability Polymerase Chain Reaction (RV-PCR) Protocol for Detection of Francisella Tularensis in Water Samples
Citation:
Shah, S., S. Kane, AND T. Alfaro. Optimization of a Rapid Viability Polymerase Chain Reaction (RV-PCR) Protocol for Detection of Francisella Tularensis in Water Samples. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-19/240, 2019.
Impact/Purpose:
Francisella tularensis, the bacterial pathogen that causes tularemia, is one of the high-priority biological threat agents. During a tularemia bioterrorism incident or a natural outbreak, the water system can be contaminated. This pathogen survives for many days in water and also, amoeba found in stagnant water. It is a very slow growing pathogen and can take several days to detect using the traditional microbiological culture methods. Therefore, a need for a rapid and high-throughput analytical method was identified and the EPA ORD’s National Homeland Security Research Center (NHSRC) addressed this need by developing and optimizing a Rapid Viability PCR protocol to relatively rapidly detect F. tularensis in water. Using this method, results can be obtained in 36 hours as compared to several days by the traditional microbiological culture methods. Also, with this method, high-throughput sample analysis can be performed using a 48-well culture plate which requires very low volume of growth medium resulting in additional advantage of significantly low laboratory waste. This method is developed for use by the EPA’s Environmental Response Laboratory Network (ERLN) that includes the Water Laboratory Alliance (WLA) network managed by the EPA Office of Water. It can also be useful to other Federal Agencies’ laboratory networks for a national level need.
Description:
This is a final report for the project on Optimization of a Rapid Viability (RV) PCR Protocol for Detection of Francisella tularensis in water. During a tularemia bioterrorism incident or a natural outbreak, the water system can be contaminated. This pathogen survives for many days in water and also, amoeba found in stagnant water. It is a very slow growing pathogen and can take several days to detect using the traditional microbiological culture methods. Therefore, a high-throughput RV-PCR protocol was developed and optimized by the EPA ORD’s National Homeland Security Research Center (NHSRC) to relatively rapidly detect F. tularensis in water. The RV-PCR method combines relatively shorter sample enrichment in growth media, and highly specific and sensitive PCR assay based detection and identification of F. tularensis. This final report describes, in detail, the R&D work done in this project, including the evaluation of the method performance in the presence of environmental interferents such as the Arizona Test Dust, iron and humic matter. Using this method, results can be obtained in 36 hours as compared to several days by the traditional microbiological culture methods.