title: Virtual Barcoding using LATE-PCR and Lights-On/Lights-Off probes: identification of nematode species in a closed-tube reaction.
abstract: The present study describes a rapid, universal, easy-to-use, closed-tube, non-sequencing method that should also be able to uniquely identify almost any animal species on earth. The approach, called Virtual Barcoding, is illustrated using five species of nematodes from three genera. Linear-After-The-Exponential (LATE) PCR was used to amplify a portion of the CO1 gene for each of five commercially available, beneficial species of soil nematodes. A set of ten low temperature Lights-On/Lights-Off consensus probes were included in the reaction mixture and were used at end-point to coat the accumulated single-stranded amplicon by dropping the temperature. Because each of the probes is mis-match tolerant, the temperature at which it hybridizes to its complementary region within the target is sequence dependent. As anticipated, each species had its own unique fluorescent signature in either three different colors, or a single color depending on which fluorophores were used to label the Lights-On probes. Each fluorescent signature was then mathematically converted to a species-specific Virtual Barcode.
authors.affiliation: a Department of Biology , Brandeis University , Waltham , MA , USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Rice
authors.affiliation: a Department of Biology , Brandeis University , Waltham , MA , USA.
authors.forename: Arthur H
authors.initials: AH
authors.lastname: Reis
authors.affiliation: a Department of Biology , Brandeis University , Waltham , MA , USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D000818:Animals; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D050259:Genes, Mitochondrial; D049751:Genome, Helminth; D009348:Nematoda / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D005456:Fluorescent Dyes
keywords: Barcode of life; CO1 gene of mitochondria; LATE-PCR; Lights-On/Lights-Off probes; Virtual Barcoding; nematodes; parasites; species identification