Simultaneous detection and discrimination of Pratylenchus and Meloidogyne species in Japanese fields using group-specific primers and denaturing gradient gel electrophoresis
2015
Kushida, A. ((NARO Hokkaido Agricultural Research Center, Kasai-gun, Hokkaido (Japan). Upland Farming Research Division), (Hokkaido University, Sapporo, Hokkaido (Japan). Graduate School of Agriculture)) | Kondo, N.
We developed a simultaneous detection and discrimination method for the main Pratylenchus and Meloidogyne species of nematodes inhabiting Japanese fields. The method consists of a single polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) process using group-specific primers (SNem 1-4) constructed from the 18S rDNA region with high specificity toward these nematodes. Optimal electrophoresis conditions to differentiate PCR products of each species were 60 degC at a constant voltage of 70 V for 17 h using DGGE gel, with a 6 % acrylamide concentration and a denaturant gradient ranging from 25 to 41 %. Differentiation of most species was achieved according to their main band positions. Only bands derived from the Pratylenchus and Meloidogyne species were present in the DGGE profiles of many nematode communities from various locations in Japan, so we concluded that these primers were sufficiently specific to the targeted nematodes. Detection sensitivity was so high that we could detect one second-stage juvenile of Pratylenchus species among 1,000 free-living nematodes using a single PCR step (35 cycles). Even when two or more target species coexisted in a community, we could usually detect and distinguish them accurately. Most target species formed some minor bands in addition to the primary band. Any interference of the minor bands with the main bands of other target species was overcome using optimal denaturing gradient conditions for the electrophoresis.
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