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.
Highlights
Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available
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 molecular taxonomic identifications performed in this study confirmed that the nematodes discovered in Beech leaf disease (BLD) leaves from Ohio and Pennsylvania are the same species of Litylenchus crenatae mccannii, but they demonstrated a technical improvement to achieve consistent amplification of the 3.5 kb ribosomal PCR product through long segment PCR amplification using sometimes variable quality crude genomic DNA extracts as template
Summary
Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. This report describes a significant technical improvement beyond previous efforts (Carta and Li, 2018, 2019) to more reliably amplify the 3.5 kb long rDNA target and increase the PCR yield for the crude, unpurified DNA extracts of single nematodes by utilizing proofreading DNA polymerase in an optimized solution. This is important because it is impractical in a nematode diagnostic laboratory to efficiently produce very clean DNA with a kit from only one or a few specimens. Two Taq-based blend systems, TaKaRa Ex Taq® DNA Polymerase (a blend of TaKaRa Taq® DNA Polymerase and an unspecified proofreading DNA polymerase) and PicoMaxxTM High Fidelity PCR System (a blend of Taq2000TM DNA polymerase, cloned Pfu DNA polymerase and ArchaeMaxx® polymerase enhancing factor) were selected and tested in this study
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