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

Abstract

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 speciesspecific 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.

Evaluation of Soil Suppressiveness of Various Japanese Soils against the Soybean Cyst Nematode Heterodera glycines and Its Relation with the Soil Chemical and Biological Properties

“…Soil DNA was extracted in duplicate with the method described by Goto et al [35]. The soil (0.5 g) was homogenized with a ball mill (FastPrep-24: Funakoshi Co., Ltd., Tokyo, Japan) and was put into a 2 mL tube including 500 µL of 20% skim milk, 0.75 g zirconia beads (0.1 mm diameter) and 0.25 g glass beads (0.5 mm diameter), mixed thoroughly and centrifuged (12,000× g for 1 min, 25 • C).…”

Development and validation of four TaqMan real-time PCR diagnostics for the identification and quantification of Pratylenchus crenatus, Pratylenchus neglectus, Pratylenchus penetrans and Pratylenchus thornei

“…While TaqMan real-time PCR offers a highly specific diagnostic, there are known limitations including false-positive reactions, DNA fragments from unknown species, or false-negative reactions due to intra-individual variation (Schrader et al, 2012). Several real-time PCR protocols for quantitative identification of several RLN species have been developed including for P. crenatus (Oliveira et al, 2017), P. penetrans (Sato et al, 2007(Sato et al, , 2010Yan et al, 2008;Goto et al, 2011;Mokrini et al, 2013;Oliveira et al, 2017;Bandoo et al, 2017;Dauphinais et al, 2018), P. thornei (Yan et al, 2012;Mokrini et al, 2014;Lin et al, 2020), P. neglectus (Yan et al, 2008(Yan et al, , 2013Oliveira et al, 2017;Lin et al, 2020), P. vulnus (Fanelli et al, 2014), P. scribneri (Arora et al, 2020) and P. zeae (Berry et al 2008). The D2-D3 expansion segment of the large subunit 28S rDNA has previously been selected as a molecular marker for phylogenetic relationships to distinguish between closely related species of Pratylenchus giving a better resolution for species separation (Al-Banna et al, 1997, 2004De Luca et al, 2004Subbotin et al, 2008;Janssen et al, 2017 a, b).…”