Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/1999
Publication Date: N/A
Citation: Interpretive Summary: Although few microorganisms are now commercially available for use against insect pests, a large group of organisms (nematodes, fungi, bacteria, rickettsia, virus and protozoa) can be significant factors in regulating insect populations under natural conditions. One group of microorganisms, the entomopathogenic nematodes or insect parasitic nematodes, show real promise for controlling many pest insects. New species, strains and isolates of entomopathogenic nematodes are being discovered throughout the world. However, the identification of these new nematode isolates, and their relationship to previously known entomopathogenic nematodes, has been problematical. We have developed a technique to give a more rapid and precise identification of nematode strains. This system, a PhastSystem PAGE gel, is commercially available and easily standardized world wide. Use of this new tool will enable scientists to more quickly and accurately identify the nematodes they find and will enable researchers to better understand the relationships between various entomopathogenic nematode species, genera and families.
Technical Abstract: A relatively rapid and economic way of identifying and assigning nematodes to taxons (which had already been determined by comparative sequence analysis of nuclear rDNA ITS region) is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem PAGE analysis of RFLP patterns of 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. The down scaling from conventional agarose to PhastSystem gels resulted in patterns 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, such as isolates from Hungary and Ireland and to provide a method of identification of previously unclassified strains.