A method for extracting genomic DNA from individual elaphostrongyline (Nematoda: Protostrongylidae) larvae and differentiation of Elaphostrongylus spp. from Parelaphostrongylus spp. by PCR assay.
This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.
Department of Biology, University of Saskatchewan, Saskatoon, Canada.
Neil B
NB
Chilton
Florence
F
Huby-Chilton
Murray W
MW
Lankester
Alvin A
AA
Gajadhar
D000818:Animals; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D049751:Genome, Helminth / Q000235:genetics*; D007814:Larva / Q000235:genetics*; D009348:Nematoda / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013045:Species Specificity
D018509:DNA, Helminth