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.

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

“…Previous results found that the sensitivity of the LAMP assay was higher than conventional PCR9262728. In the present study, the detection limit was low to 1/200 000 nematode DNA, whereas the conventional PCR approach usually requires a single nematode, Williamson et al 11.…”

“…Traditional M. hapla detection assays based on PCR methods1112131530 are time-consuming and require sophisticated equipments as well as expertise. LAMP is a novel, simple, rapid and precise amplification method that can be adapted for diagnosing plant-parasitic nematodes, as previously reported925262728. In this study, we designed a set of five specific LAMP primers to detect M. hapla .…”

“…Therefore, the detection limit of the LAMP was 100 times higher than that of PCR-based detection methods. The sensitivity of the LAMP was equal to B. xylophilus 25, M. enterolobii 9, M. incognita 26, R. similis 27 and T. semipenetrans 28.…”

“…The direct detection of plant nematodes using DNA from infected plant tissues has been published2731323334, although those methods for preparing nucleic acids require toxic reagents and expansive instruments. In this study, direct extraction of DNA from plant root galls greatly improved the efficiency when using the FTA technology.…”

“…Because it is high efficient and does not require special laboratory facilities, this assay has recently shown promising in the specific and rapid detection of clinical2021 and agricultural pathogens222324. However, in plant parasitic nematodes, only Bursaphelenchus xylophilus 25, M. enterolobii 9, M. incognita 26 Radopholus similis 27 and Tylenchulus semipenetrans 28 have been detected by LAMP. In this study, we established a sensitive and specific LAMP method for the direct detection of M. hapla from infected plant root galls based on the rDNA-ITS.…”