Using DNA suspension arrays to identify library-independent markers for bacterial source tracking.
We developed a suspension array to enhance the ability to use library-independent genetic markers for bacterial source tracking. Six markers from Enterococcus spp. were selected to distinguish between cattle, humans, and cervids. Multiplex PCR was used to amplify fecal markers and resulting products were biotinylated and fragmented by nick translation followed by hybridization to polystyrene beads. Six populations of beads were included simultaneously in each assay where beads were labeled with an oligonucleotide probe complementary to one of the six library-independent markers. Hybridized products were detected on the beads using a 2-laser flow cytometer in a 96-well format. Testing with previously characterized strains showed that the assay could achieve 100% diagnostic sensitivity and >95% diagnostic specificity. Results from water samples were congruent for conventional PCR. Serial dilutions of template DNA demonstrated that the bench top analytic sensitivity of the entire assay was equivalent to <1600 cells. Suspension arrays permit greater certainty of product identification and this format can be expanded to include many additional markers.
Call
Douglas R
DR
Department of Veterinary Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164 7040, USA. drcall@wsu.edu
Satterwhite
Dennis M
DM
Soule
Marilyn
M
D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods / Q000592:standards; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D016983:Enterococcus / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D005618:Fresh Water / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D013045:Species Specificity; D014876:Water Pollution / Q000032:analysis*
D004269:DNA, Bacterial; D005819:Genetic Markers