2005
DOI: 10.1177/104063870501700612
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A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

Abstract: Abstract. 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 detecte… Show more

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Cited by 5 publications
(7 citation statements)
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“…With the exception of the island of Newfoundland, where Elaphostrongylus rangiferi Mitskevich, 1960 is thought to have been introduced along with reindeer (Rangifer tarandus tarandus (L., 1758)) from the Old World, DSL in either woodland (Rangifer tarandus caribou (Gmelin, 1788)) or barrenground (Rangifer tarandus groenlandicus (Borowski, 1780) and Rangifer tarandus grantii (Allen, 1902)) caribou native to North America have been assumed to be Parela-phostrongylus andersoni Prestwood, 1972or Parelaphostrongylus odocoilei (Hobmaier and Hobmaier, 1934) (e.g., Lankester 2001). In the Canadian north, the elaphostrongylines P. andersoni and P. odocoilei have been confirmed previously in woodland caribou and P. andersoni in barrenground caribou by postmortem recovery and morphological examination of adult nematodes (Lankester and Hauta 1989) or by molecular analyses of larvae (Jenkins et al 2005a;Chilton et al 2005, Huby-Chilton et al 2006. Unidentified DSL were found previously in woodland caribou from the Mealy Mountains, Labrador (Lankester and Hauta 1989), from northeastern Alberta (Gray and Samuel 1986), and from Manitoba and Ontario (Lankester et al 1976).…”
Section: Introductionsupporting
confidence: 59%
“…With the exception of the island of Newfoundland, where Elaphostrongylus rangiferi Mitskevich, 1960 is thought to have been introduced along with reindeer (Rangifer tarandus tarandus (L., 1758)) from the Old World, DSL in either woodland (Rangifer tarandus caribou (Gmelin, 1788)) or barrenground (Rangifer tarandus groenlandicus (Borowski, 1780) and Rangifer tarandus grantii (Allen, 1902)) caribou native to North America have been assumed to be Parela-phostrongylus andersoni Prestwood, 1972or Parelaphostrongylus odocoilei (Hobmaier and Hobmaier, 1934) (e.g., Lankester 2001). In the Canadian north, the elaphostrongylines P. andersoni and P. odocoilei have been confirmed previously in woodland caribou and P. andersoni in barrenground caribou by postmortem recovery and morphological examination of adult nematodes (Lankester and Hauta 1989) or by molecular analyses of larvae (Jenkins et al 2005a;Chilton et al 2005, Huby-Chilton et al 2006. Unidentified DSL were found previously in woodland caribou from the Mealy Mountains, Labrador (Lankester and Hauta 1989), from northeastern Alberta (Gray and Samuel 1986), and from Manitoba and Ontario (Lankester et al 1976).…”
Section: Introductionsupporting
confidence: 59%
“…For example, in North America, adult nematodes of five genera within the family Protostrongylidae (Elaphostrongylus, Muellerius, Parelaphostrongylus, Umingmakstrongylus, and Varestrongylus) produce first-stage larvae with a dorsal-spine on the tail (DSL; Anderson, 2000), which cannot be distinguished from one another using morphologic characters (Lankester, 2001). Thus, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analyses, and DNA sequencing have been used to determine the species identity of DSL based on sequence differences in the first or second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal (r) DNA (Gajadhar et al, 2000;Chilton et al, 2005;Jenkins et al, 2005;Huby-Chilton et al, 2006a, b;Mortenson et al, 2006;Kutz et al, 2007;Asmundsson et al, 2008).…”
mentioning
confidence: 99%
“…The identification of protostrongylid DSL to the species or genus level based on geographic location and host species is not feasible because of the possibility of mixed infections and overlapping host distributions (Pybus and Samuel, 1981;Lankester and Fong, 1998). Moreover, as DSL of different elaphostrongyline species are morphologically and morphometrically similar (Lankester and Hauta, 1989;Gajadhar, et al, 1994), only molecular studies have been used effectively for identification of species (Gajadhar et al, 2000;Chilton et al, 2005;Jenkins et al, 2005a;Huby-Chilton et al, 2006). The DNA sequencing of the second internal transcribed spacer (ITS-2) rDNA has been used in species identification and for determining the geographic distribution and host range of P. odocoilei (see Jenkins et al, 2005a).…”
mentioning
confidence: 99%
“…For the molecular analyses, 10 ethanol-fixed DSL were washed three times in sterile nanopure water. Genomic (g) DNA was isolated from individual DSL by sodium dodecyl-sulfate/proteinase K treatment and then column-purified using the Wizard TM DNA CleanUp kit (Promega, Madison, Wisconsin, USA) (Chilton et al, 2005). A partial fragment (317-336 bp) of the ITS-1 rDNA and 59 flanking region (76 bp of the 18S rRNA gene) was amplified by PCR from 2 ml of gDNA using the oligonucleotide primers NC16 (forward, 59-AGT TCA ATC GCA ATG GCT T-39) and R1 (reverse, 59-GCA TTC TAG CAA TGC TCA TT-39) and the conditions described by Huby- Chilton et al (2006).…”
mentioning
confidence: 99%