Loop-mediated isothermal amplification for rapid and precise detection of the burrowing nematode, Radopholus similis, directly from diseased plant tissues

Abstract

A novel, simple, rapid and highly sensitive assay and diagnostic tool for the burrowing nematode, Radopholus similis, was developed using a loop-mediated isothermal amplification (LAMP). The LAMP assay was targeted on the specific fragments of rRNA gene D2-D3 regions of R. similis. The detection limitation of the LAMP assay was as low as ten copies of plasmid DNA containing the target DNA, 10 fg of genomic DNA and 5 × 10−5 nematodes. The detection sensitivity of the LAMP method for R. similis DNA was 10-100 times higher than normal PCR-based detection methods. The LAMP amplifications could be observed directly by eye by adding SYBR Green I and the lateral flow dipstick (LFD). LAMP assay for R. similis is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by R. similis. The LAMP assay developed in this study is highly effective, easy to perform and readily adaptable for diagnostic and monitoring of the R. similis-diseased seedling in the field.