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.
Chilton
Neil B
NB
Department of Biology, University of Saskatchewan, Saskatoon, Canada.
Huby-Chilton
Florence
F
Lankester
Murray W
MW
Gajadhar
Alvin A
AA
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