Application of a simple and high-throughput DNA extraction method to real-time PCR quantification of target plant-parasitic nematodes in nematode communities

Abstract

Molecular techniques are prevailing in nematode identification and quantification, for which DNA extraction from nematodes is essential. However, the published DNA extraction methods are laborious and often require various expensive consumables and high-end equipment. In order to prepare DNA templates for a high-throughput real-time PCR assay, the present study modified a conventional DNA extraction method of Naklha et al. (2010) from nematode suspensions with ordinary lab equipment and achieved results and advantages comparable with two other conventional methods. The results of real-time PCR assays for quantifying Pratylenchus zeae, Tylenchorhynchus leviterminalis and Hoplolaimus sp. using the new protocol were highly correlated with those obtained by morphological counts and comparable to or more sensitive than those obtained by two conventional methods. Although the new protocol took over 100 min for DNA extraction, the manual processing took less than 10 min, i.e., half to one-fourth of the other methods. The running cost was less than half to one-tenth of the other methods.