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    Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes


    Pamjav, Horolma and Triga, Dimitra and Busaz, Zsuzsanna and Vellai, Tibor and Lucskai, Attila and Adams, Byron and Reid, Alexander P. and Burnell, Ann and Griffin, Christine and Glazer, Itamar and Klein, Michael G. and Fodor, Andras (1999) Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes. Electrophoresis, 20 (6). pp. 1266-1273. ISSN 0173-0835

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    Abstract

    A relatively rapid and economic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)-amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272—1277). The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis “Irish Type”, represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

    Item Type: Article
    Keywords: Molecular diagnostics; identification; entomopathogenic; heterorhabitics; Steinernema;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7548
    Identification Number: https://doi.org/10.1002/(SICI)1522-2683(19990101)20:6<1266::AID-ELPS1266>3.0.CO;2-4
    Depositing User: Dr. Christine Griffin
    Date Deposited: 19 Oct 2016 14:43
    Journal or Publication Title: Electrophoresis
    Publisher: Wiley-VCH Verlag
    Refereed: Yes
    URI:

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