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

Authors

Horolma Pamjav, Eötvös University, Budapest, Hungary
Dimitra Triga, Eötvös University, Budapest, Hungary
Zsuzsanna Buzµs, Agricultural Biotechnology Center, Gödöllð, Hungary
Tibor Vellai, Eötvös University, Budapest, Hungary
Attila Lucskai, Institute of Plant Protection, Georgikon Agricultural Faculty, Pannon University of Agricultural Sciences, Keszthely, Hungary
Byron Adams, University of Nebraska-LincolnFollow
Alexander P. Reid, CAB International, International Institute of Parasitology, St. Albans, Herts, UK
Ann Burnell, University of Ireland, Maynooth, Ireland
Christine Griffin, University of Ireland, Maynooth, Ireland
Itamar Glazer, A.R.O. The Volcani Center, Bet Dagan, Israel
Michael G. Klein, USDA
Andras Fodor, Eötvös University, Budapest, Hungary

Date of this Version

5-26-1999

Comments

Published in Electrophoresis 1999, 20, 1266±1273.

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 ªIrish Typeº, 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.