Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, Meloidogyne hapla, Using Recombinase Polymerase Amplification Assays.

Sergei A Subbotin,Julie Burbridge

doi:10.3390/plants10020336

Abstract

Rapid and reliable diagnostics of root-knot nematodes are critical for selections of effective control against these agricultural pests. In this study, recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the northern root-knot nematode, Meloidogyne hapla. The RPA assays using TwistAmp® Basic, TwistAmp® exo and TwistAmp® nfo kits (TwistDx, Cambridge, UK) allowed for the detection of M. hapla from crude extracts of females, eggs and juveniles without a DNA extraction step. The results of the RPA assays using real-time fluorescence detection (real-time RPA) in series of crude nematode extracts showed reliable detection after 13 min with a sensitivity of 1/100 of a second-stage juvenile and up to 1/1000 of a female in reaction tubes. The results of the RPA assays using lateral flow dipsticks (LF-RPA) showed reliable detection within 30 min with a sensitivity of 1/10 of a second-stage juvenile and 1/1000 of a female in reaction tubes. The RPA assay developed here is a successful tool for quick, accurate and sensitive diagnostics of M. hapla. The application of the LF-RPA assay has great potential for diagnosing infestation of this species in the lab, field or in areas with a minimal laboratory infrastructure.

Highlights

The northern root-knot nematode, Meloidogyne hapla is one of the four most common root-knot nematode species worldwide
We have developed an affordable, simple, fast and sensitive real-time recombinase polymerase amplification (RPA) and LF-RPA assays to detect M. hapla from nematode specimens extracted from plant and soil samples
An LF-RPA assay can be performed in a field condition without any special equipment or in areas with a minimal laboratory infrastructure

Summary

Introduction

The northern root-knot nematode, Meloidogyne hapla is one of the four most common root-knot nematode species worldwide. This nematode is extremely polyphagous, attacking a wide variety of crops and weeds. Several authors developed a TaqMan real-time PCR assay with species-specific primers for the detection of M. hapla from root galls and soil samples [8,9,10,11,12]. Peng et al [13] developed loop-mediated isothermal amplification methods (LAMP) combined with a Flinders Technology Associates card for the identification of M. hapla

Methods

Results

Conclusion