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Rapid and sensitive detection of Meloidogyne mali by loop-mediated isothermal amplification combined with a lateral flow dipstick

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Abstract

Meloidogyne mali Itoh, Ohshima & Ichinohe, 1969 is a polyphagous root-knot nematode that infects a wide range of host plants and is subject to phytosanitary restrictions in many countries, including China. In this study, we integrated the loop-mediated isothermal amplification (LAMP) technique with visual detection by a lateral flow dipstick (LFD) and developed an accelerated LAMP-LFD method for the rapid identification of M. mali, targeting the internal transcribed spacer regions and 5.8S ribosomal DNA sequence (ITS-5.8S). The entire test could be performed within 45 min, including 35-min LAMP amplification, 5-min hybridization, and 3–5-min visualization on LFD. The detection limit of this method was 2-pg bulked M. mali gDNA per reaction, and the equivalent of 1‰ single female adult or 1% single second-stage juvenile (J2) could be detected. It is recommended that DNA is extracted from single J2 isolated from soil samples by the modified Baermann funnel method or from an adult female from galls. Based on this, the field sensitivity and specificity of this LAMP-LFD method for the detection of M. mali in field soil samples were both 100% compared to traditional microscopic observation. These results indicate that the LAMP-LFD method is rapid, sensitive, accurate, and simple, and would be applicable for the routine monitoring of phytosanitary M. mali.

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Acknowledgements

The project was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA092001), Zhejiang Provincial Natural Science Foundation of China (No. LY14C180002), the Scientific Innovation Team Project of Ningbo (No. 2015C110018) and the KC Wong Magna Fund in Ningbo University.

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Authors and Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo, 315211, China

    Qian-jin Zhou, Yi Cai & Jiong Chen

  2. Ningbo Academy of Inspection and Quarantine, Ningbo, Zhejiang, 315012, China

    Jian-feng Gu

  3. College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China

    Xuan Wang

  4. The Donghai Sea Collaborative Innovation Center for Industrial Upgrading Mariculture, Ningbo University, Ningbo, 315211, China

    Jiong Chen

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  1. Qian-jin Zhou
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  2. Yi Cai
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Correspondence to Jiong Chen.

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Zhou, Qj., Cai, Y., Gu, Jf. et al. Rapid and sensitive detection of Meloidogyne mali by loop-mediated isothermal amplification combined with a lateral flow dipstick. Eur J Plant Pathol 148, 755–769 (2017). https://doi.org/10.1007/s10658-016-1130-4

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