A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

• Published in: Journal of Veterinary Diagnostic Investigation
• Main Authors: Chilton, Neil B., Huby-Chilton, Florence, Lankester, Murray W., Gajadhar, Alvin A.
• Format: Article in Journal/Newspaper
• Language: English
• Published: SAGE Publications 2005
• Subjects: General Veterinary; Canada; Newfoundland
• Online Access: http://dx.doi.org/10.1177/104063870501700612; http://journals.sagepub.com/doi/pdf/10.1177/104063870501700612

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L 1 ) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L 1 s 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 L 1 s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L 1 s, 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.