Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease.

Abstract

Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.