Q-261. Enumeration and Differentiation of Bacteroides Isolates Using a Dual-Probe, Quantitative Polymerase Chain Reaction (qPCR) Coupled with Melt Analysis

R. A. Reinke;
Sanitation Districts of Los Angeles County, Whittier, CA.

Microbial source tracking studies often rely on bacterial pattern recognition and correlation to known indicators to help identify potential sources of contamination. Bacteroides species have been extensively used in microbial source tracking studies but cross-reactivity with non-source specific isolates has been reported. To address this, two real-time qPCR assays have been developed that target total Bacteroides or human-specific Bacteroides species. The use of dual-hybridization probe chemistry allows melt analyses to be performed following the qPCR. This is used to detect genetic differences within the probe binding sites yielding an additional level of specificity. Bacteroides species containing differing numbers of mutations within the probe binding sites were tested to determine the limit of detection (LOD) for the assay as well as the potential to differentiate between isolates based on melt analysis. The results demonstrate that the human-specific assay has a LOD on the order of 100 to 102 colony forming units (CFU) for isolates containing zero to two nucleotide changes. The total Bacteroides assay demonstrated an LOD on the order of 100 CFU. Furthermore, plasmid encoded amplicons were used to determine the LOD in genomic equivalent copies and resulted in LODs on the order of 101 copies for both assays. Melt analysis performed on amplicons from the human-specific Bacteroides assay show that isolates containing zero, one or two changes within the probe sequence can be distinguished with melt temperatures of 67oC, 65oC, and 60oC respectively. To examine the cross-reactivity potential both assays were tested against more than 110 different isolates representing over 37 different species. No cross-reactivity was observed. The qPCR described herein offers significant advantages over traditional PCR methods of Bacteroides detection. In particular, the assay can be used to quantitatively track both total and human-specific Bacteroides species throughout a project site as well as identify and track genotypically different Bacteroides isolates in a single sample by melt curve analysis.