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Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei

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Abstract

Pratylenchus neglectus and P. thornei are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the P. neglectus-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the P. thornei-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other Pratylenchus species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from P. neglectus and P. thornei DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (Ct) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of P. neglectus and P. thornei.

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Acknowledgements

This work was supported by the Planning Project for Forestry Science and Technology in Guangdong Province (grant number 2015KJCX045) and the Planning Project for Science and Technology in Guangzhou City (grant number 11A62100574).

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Authors and Affiliations

  1. Laboratory of Plant Nematology, South China Agricultural University, Guangzhou, 510642, China

    Borong Lin, Ye Tao, Jinling Liao & Kan Zhuo

  2. Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou, 510642, China

    Borong Lin, Ye Tao, Jinling Liao & Kan Zhuo

  3. Guangdong Eco-Engineering Polytechnic, Guangzhou, 510520, China

    Honghong Wang & Jinling Liao

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  1. Borong Lin
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  2. Ye Tao
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  3. Honghong Wang
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  4. Jinling Liao
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  5. Kan Zhuo
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Correspondence to Kan Zhuo.

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Lin, B., Tao, Y., Wang, H. et al. Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei. Eur J Plant Pathol 157, 185–196 (2020). https://doi.org/10.1007/s10658-020-01999-7

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  • DOI: https://doi.org/10.1007/s10658-020-01999-7

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