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Improved HF183 reverse primer and probe for greater analytical sensitivity of human Bacteroides in the environment
Citation:
Millen, H., S. Spencer, O. Shanks, H. Green, C. Kelty, Rich Haugland, J. Gonnering, AND M. Borchardt. Improved HF183 reverse primer and probe for greater analytical sensitivity of human Bacteroides in the environment. Presented at American Society for Microbiology 2013, Denver, CO, May 18 - 21, 2013.
Impact/Purpose:
To inform the public.
Description:
Background: Numerous indicators have been used to assess the presence of fecal pollution, many relying on molecular methods such as qPCR. One of the targets frequently used, the human-associated Bacteroides 16s rRNA region, has several assays in current usage. These assays vary in terms of target specificity and analytical sensitivity, which makes accurate estimation difficult in environmental samples, like groundwater, where human Bacteroides concentrations are typically low and other fecal microbes are present. Methods: Environmental samples were taken from groundwater and surface water using glass wool filtration and from wastewater using 1L grab samples. These were further concentrated by PEG precipitation. Samples were analyzed with two Bacteroides 16S gene assays, an existing human-associated HF183 Bacteroides assay and HuBac. Modifications were made to the reverse primer and probe to optimize the HF183 assay. The existing and improved HF183 assays were evaluated for non-target amplification, sensitivity and specificity using SYBR melt curves, gel electrophoresis, and dilution series of human fecal samples in the environmental concentrates. Results: The existing human Bacteroides HF183 assay and HuBac were found to have non-target amplification, precluding accurate detection of human fecal material at low concentrations. The excessive primer-dimer formed by the existing HF183 assay caused a reduction in fluorescence plateau height due to limiting reagents for target amplification. With the existing HF183 assay, a dilution series performed in triplicate showed the plateau height (fluorescence units at 45 cycles) was reduced 87.73% between 0.1ng of untreated sewage (mean Ct value of 28.05) and .001ng of untreated sewage (mean Ct value of 34.5). Modification of the HF183 reverse primer and probe decreased primer-dimer formation, as evidenced by the plateau height decreasing by only 7.69% between the 0.1ng dilution (mean Ct of 27.98) and the .001ng dilution (mean Ct of 35.03). Conclusion: Assay design, optimization, and rigorous testing are important before using qPCR to assess the low target concentrations frequently found in the environment. Here, we present a modification to a human-specific Bacteroides assay which is useful to determine fecal pollution in water samples.
