Recombinase polymerase amplification assay for rapid detection of the root-knot nematode Meloidogyne enterolobii

Summary Rapid diagnosis tools for detection of root-knot nematodes play an important role in the disease control and eradication programme. Recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the pacara earpod tree root-knot nematode, Meloidogyne enterolobii. The RPA assays using TwistAmp® Basic and TwistAmp® exo kits allowed detection of M. enterolobii from gall tissues and crude nematode extracts of all stages of target species without a DNA extraction step. The results of real-time RPA assays using a real-time fluorescent detection of a series of crude nematode extracts showed reliable detection with sensitivity of 1/10 of a second-stage juvenile in a RPA reaction tube after 15-20 min. The RPA assay provides affordable, simple, fast and sensitive detection of M. enterolobii.

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

Plants ◽

10.3390/plants10020336 ◽

2021 ◽

Vol 10 (2) ◽

pp. 336

Author(s):

Sergei A. Subbotin ◽

Julie Burbridge

Keyword(s):

Real Time ◽

Recombinase Polymerase Amplification ◽

Effective Control ◽

Rrna Gene ◽

Meloidogyne Hapla ◽

Agricultural Pests ◽

Root Knot Nematode ◽

Second Stage ◽

Reliable Detection ◽

Stage Juvenile

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.

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Evaluation of Recombinase Polymerase Amplification Assay for Detecting Meloidogyne javanica

Plant Disease ◽

10.1094/pdis-07-19-1473-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 801-807

Author(s):

Yuan-kai Chi ◽

Wei Zhao ◽

Meng-di Ye ◽

Farman Ali ◽

Tao Wang ◽

...

Keyword(s):

Adult Female ◽

Marker Gene ◽

Gene Segment ◽

Meloidogyne Javanica ◽

Recombinase Polymerase Amplification ◽

Lateral Flow Dipstick ◽

Amplification Assay ◽

Stage Juvenile ◽

Sequence Characterized Amplified Regions ◽

Recombinase Polymerase Amplification Assay

Meloidogyne javanica is one of the most widespread and economically important nematodes in many countries, including China. In this study, a recombinase polymerase amplification (RPA) assay was evaluated for the detection of M. javanica based on the sequences of a sequence-characterized amplified regions marker gene segment. The RPA assay specifically detected M. javanica from individual juvenile or adult female, M. javanica-induced galls, and nematodes in the soil samples. The detection limit of M. javanica RPA assay was 1 pg of purified genomic DNA, 0.01 adult female, or 0.1 second-stage juvenile, which was 10 times more sensitive than conventional PCR assay. Furthermore, combined with lateral flow dipstick (LFD), a visual detection method of LFD-RPA assay was developed, which is suitable for onsite surveys and routine diagnostics. Results indicate that the RPA assay is rapid, sensitive, and reliable for detection and molecular identification of M. javanica.

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