Identification of the potato cyst nematodes based on two-step multiplex endpoint PCR with the dUTP/UNG system for carry-over prevention
2019
Sakai, H. (Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Sapporo, Hokkaido (Japan)) | Kushida, A. | Narabu, T.
Multiplex endpoint PCR techniques are among the essential diagnostic tools used for identifying the potato cyst nematodes (PCNs: Globodera rostochiensis and G. pallida). Multiplex endpoint PCR assays for PCNs published to date, based on three-step PCR, are not integrated with primers to verify successful PCR in testing non-target species. Besides, carry-over contamination is a serious problem in diagnostic PCR assays. To improve the run time and reliability of the endpoint PCR test, we developed a two-step multiplex PCR identification method for PCNs using the dUTP/UNG carry-over prevention system. For this purpose, primers with high melting temperatures were newly designed to amplify mitochondrial DNA fragments specific to the respective PCN species and the nuclear ribosomal RNA gene fragments as PCR positive controls across cyst nematodes. In addition, the DNA preparation method from juveniles and cysts was simplified using sodium dodecyl sulfate and disposable homogenizers. This multiplex amplification generated amplicons of 150 bp, 287 bp and ca. 450 bp for G. rostochiensis, G. pallida and the non-target cyst nematode species, respectively. No cross-reactions were observed among the tested nematodes including 11 species and 22 populations. In examining mixed juveniles and cysts of PCNs, our method successfully detected both species even in the ratio of one to ten. These PCR runs took ca. 1 h, faster than the other three-step PCR protocols. The new PCR diagnostic method described here is reliable, fast and thus a good alternative to the other assays based on conventional PCR for diagnosis of PCNs.
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