ARS | Publication request: Single laboratory comparison of host-specific PCR assays for the detection of bovine fecal pollution

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2009
Publication Date: May 19, 2009
Citation: Shanks, O., White, K., Kelty, C., Hayes, S., Sivaganesan, M., Jenkins, M., Varma, M., Haugland, R. 2009. Single laboratory comparison of host-specific PCR assays for the detection of bovine fecal pollution [abstract]. American Society for Microbiology.

Technical Abstract: There are numerous PCR-based methods available to detect bovine fecal pollution in ambient waters. Each method targets a different gene and microorganism leading to differences in method performance, making it difficult to determine which approach is most suitable for field applications. We describe the performance assessment of seven PCR and qPCR assays reported to be specific for either ruminant or bovine feces. Each assay was tested against a reference collection of 247 individual bovine fecal samples representing 11 different populations and 184 fecal DNA extracts from 30 different animal species. Previously reported host-specific assays demonstrated a broad distribution among individual bovine samples (38.5-93.2%) and exhibited specificity levels ranging from 54.1% to 100%. End-point PCR sensitivity testing at four different target DNA concentrations indicated a 100% sensitivity level at a 1x10-9 g of target DNA concentration and 0% at 1x10-12 g. For quantitative real-time PCR assays, the abundance of each host-specific genetic marker was measured within each bovine population and compared to quantities of traditional general fecal indicator bacteria determined by real-time 16S rRNA PCR assays specific for total Bacteroidetes, Clostridia, and enterococci fecal microorganisms. Comparison experiments indicate a large range of assay specificities and also demonstrate substantial differences in sensitivity and/or abundance of genetic markers between bovine populations suggesting that some assays may be more suitable for microbial source tracking applications.