Comparison of Four Polymerase Chain Reaction Methods for the Rapid Detection of Human Fecal Pollution in Marine and Inland Waters

Abstract

We compared the effectiveness of three PCR protocols for the detection of Bifidobacterium adolescentis and one PCR protocol for detecting Bacteroidales as indicators of human fecal pollution in environmental samples. Quantitative PCR indicated that a higher concentration of B. adolescentis DNA was recovered from sewage samples on the 0.2 μm filters compared to the 0.45 μm filters, and there was no evidence of qPCR inhibitors in the DNA extracts. With the Matsuki method (1999), B. adolescentis was detected only in undiluted sewage samples. The King method (2007) performed well and detected B. adolescentis in all of the sewage dilutions (from undiluted to 10−4). In contrast, the Bonjoch approach (2004) was effective at detecting B. adolescentis at lower dilutions (10−3) of sewage samples and it gave false positive results with some (3/8) pig fecal samples. Human-specific Bacteroidales (HuBacs) were detected in the lower diluents of sewage samples but was positive in pig (6/8) and cattle fecal samples. PCR detection of B. adolescentis in marine samples from Puerto Rico and freshwater samples from Georgia indicated that the PCR method of King et al. (2007) and the modified Layton method for HuBac were in agreement in detecting human fecal pollution in most sites.

Detection of <i>Stx</i>2 Gene of <i>Escherichia coli</i> and Elevated Levels of Fecal Bacteria in the Cattle Farming Regions of Lake Oconee

“…The detection of the RuBac marker at the cattle farming sites agrees with previous MST research that demonstrated that cattle are the major source of fecal pollution in this region of the Oconee watershed [8,19,28]. Even though we did not observe cross reaction between the HuBac primers and cattle fecal DNA in our controls, it was still possible that the detection of the HuBac marker in some of the cattle farming sites could be attributed to the tendency of this PCR assay to cross react with cattle fecal samples [15,29]. This reasoning is supported by the fact that there were no houses in close proximity to the cattle farms in the watershed.…”

“…Environmental samples were assessed for possible PCR inhibition by amending with Bifidobacteria DNA as described by Bachoon et al 010. Changes of less than two C T value were observed, which indicates that the extracted DNA did not contain impurities that significantly inhibited the PCR (Bachoon et al 2010). The detection limit for STEC was 1.50 × 10 2 gene copies per 100 mL of water sample.…”

Bifidobacterial Diversity and the Development of New Microbial Source Tracking Indicators

“…porcinum has been proposed as an MST marker for animal species (35). Some of these Bifidobacterium markers have been successfully applied in Europe and some parts of the United States (2,8,14,21,26,38).…”

Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

“…In fact, the number of studies with a comprehensive temporal component is relatively small, and more importantly, most have been conducted with samples collected in temperate regions. To date, most source-tracking studies conducted in tropical waters have not comprehensively accounted for seasonal variations, have been conducted using a small number of sites, and have not evaluated the host specificity and host distribution of the assays using a significant number of fecal samples from targeted and nontargeted hosts (19,43).…”