Q-258. Bacteroidales PCR Source Tracking - Challenges and Considerations

J. A. Truesdale, N. F. Garcia, E. A. Casarez, G. D. Di Giovanni;
Texas AgriLife Research, Texas A&M Univ., El Paso, TX.

The Bacteroidales PCR assay is an emerging tool for microbial source tracking. The advantages of using Bacteroidales PCR to identify sources of fecal pollution are that it is library independent, fast and relatively easy to perform. Disadvantages include non-quantitative results, uncertain relationships to regulated fecal indicator bacteria, and a lack of standardized protocols. Several published studies have used the Bacteroidales PCR assay for source tracking with favorable results. Although these studies showed promise for Bacteroidales PCR, this same conclusion was not always obtained in other laboratories. In particular, there are significant differences in published PCR annealing temperatures and cycling conditions, even from the same researchers. For example, with the ruminant CF128 forward primer and the human HF183 forward primer the annealing temperatures had to be adjusted by as much as 4°C to reduce non-specific amplification. Obviously there is a need for additional PCR optimization and standardization. We have been using the Bacteroidales PCR assay on water and fecal samples from different locations in Texas. In order to achieve optimum PCR results for specific host primers, we are evaluating annealing temperatures using temperature gradients. In some cases it was discovered that changes to the annealing temperatures compared to published conditions greatly reduced non-specific amplification, and for other primers prevented false negatives. Still amplification of non-target animal groups has been observed by our lab and others with some primer sets, especially the CF128 ruminant primer. For example, we have experienced cross reactivity in reactions with the CF128 forward primer and fecal specimens from hogs. The use of the HF183 human specific primer has also resulted in a few false positives with animal groups such as whitetail deer and rabbit. Bacteroidales PCR has the potential to be an efficient tool for identifying non-point sources of fecal pollution. However, the sensitivity and specificity of the assay still need thorough evaluation, and standardized protocols will need to undergo multi-lab testing.