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A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan

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For more content, see Nematologica.

Multiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

Affiliations: 1: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan; 2: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan; National Agricultural Research Center for Western Region 200 Ueno, Uenocho, Ayabe, Kyoto 623-0035, Japan


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