Microplate subtractive hybridization to enrich for bacteroidales genetic markers for fecal source identification.
The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).
Dick
Linda K
LK
Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, Oregon 97331, USA.
Simonich
Michael T
MT
Field
Katharine G
KG
D000818:Animals; D041963:Bacteroidetes / Q000145:classification* / Q000235:genetics; D002417:Cattle; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004285:Dogs; D005243:Feces / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014871:Water Microbiology; D014876:Water Pollution
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005819:Genetic Markers; D012335:RNA, Ribosomal