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Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by real time PCR
Citation:
HAUGLAND, R. A., M. VARMA, M. SIVAGANESAN, C. A. KELTY, L. PEED, AND O. C. SHANKS. Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by real time PCR. SYSTEMATIC AND APPLIED MICROBIOLOGY. Elsevier Science Ltd, New York, NY, 33(6):348-357, (2010).
Impact/Purpose:
1) Develop and evaluate qPCR assays and test methods for the detection and quantification of genetic markers from indicator bacteria that are associated with human fecal waste and from two new groups of general fecal indicator bacteria (E. coli and Clostridia) that historically have been widely used or are favored in specific regions 2) Determine the occurrence and densities of genetic markers detected by new qPCR assays developed under objective 1 and compare with occurrence and densities of genetic markers detected by previously developed qPCR assays for enterococci and total Bacterioidalesin waste waters and fecal material from different animal sources. 3) Determine stability of fecal indicator bacteria target DNA sequences in freezer archived filter retentates of ambient surface water samples 4) Determine the densities of human and general fecal indicator markers in a wide range of surface and recreational waters including archived samples from previous NEEAR studies.
Description:
Molecular methods for rapidly quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted by primers for the development of real-time PCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and laboratory experimental analyses suggested that each of the assays was either fully or highly discriminatory in detecting DNA from its targeted species. Acceptable analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4 x 104 target sequence copies. The assays were applied in a preliminary assessment of the occurrence and relative abundance of their target DNA sequences in feces from humans and five other animal host groups as well as in 15 sewage influent samples from 13 different treatment facilities. Analysis results indicated that DNA targets from each of the species were present at high levels (> 103 sequence copies/ng total extracted DNA) in human wastes. Target sequences were also detected by each of the assays in all sewage samples and, with exception of Prevotella sequences, showed similar and highly correlated (R2 > 0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance of these DNA targets differed substantially in the fecal samples from the other five animal host groups. These observations may be significant in guiding future applications of these assays for identifying human fecal pollution in surface waters.