Recombinase Polymerase Amplification assays for detection of the major tropical root-knot nematodes

Subbotin, Sergei A.; Burbridge, Julie

doi:10.21307/jofnem-2021-109

Cited by 3 publications

(1 citation statement)

References 11 publications

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“…Polymerase chain reaction (PCR) is the most frequently used molecular technique in pathogen diagnosis [ 4 ]. In recent years, more advanced techniques such as real-time PCR [ 3 , 5 ], LAMP [ 6 , 7 ], RPA [ 8 , 9 ], and ddPCR [ 10 , 11 ] are also available that can give qualitative as well as quantitative measurements of the nematodes. These techniques can detect and quantify the nematodes from a pure population, mixed populations, as well as directly from total soil DNA, thus alleviating the need for nematode collection from soil and DNA extraction from nematodes.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil

Lawaju,

Yan

2024

IJMS

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This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0–3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR’s capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

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Section: Introductionmentioning

confidence: 99%

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil

Lawaju,

Yan

2024

IJMS

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Visualized Detection of Tobacco Anthracnose by a Recombinase Polymerase Amplification–Lateral Flow Dipstick Assay

Li,

Feng,

Chen

et al. 2024

Plant Disease

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Anthracnose caused by Colletotrichum spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of Colletotrichum spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main Colletotrichum spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (chs1) and could be performed in a few minutes (6–10 min). All isolates of C. kastii, C. fructicola and C. gloeosporioides yielded positive results using the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting DNA rapidly from tobacco leaves infected with C. kastii, C. fructicola and C. gloeosporioides. Based on these results, the RPA-LFD assay is easy to operate, rapid and equipment independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.

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Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia

Subbotin,

Palomares-Rius,

Castillo

2024

Journal of Nematology

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Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique that has been adopted for simple, robust, rapid, reliable diagnostics of nematodes. In this study, the real-time RPA assay and RPA assay combined with lateral flow dipsticks (LF-RPA) have been developed targeting the ITS rRNA gene of the British root-knot nematode, Meloidogyne artiellia. The assay provided specific and rapid detection of this root-knot nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.125 second-stage juvenile (J2) specimen per a reaction tube for real-time RPA during 11 min and a sensitivity of 0.5 J2 specimens per a reaction tube for LF-RPA during 25 min. The RPA assays were validated with a wide range of non-target root-knot nematodes. The LF-RPA assay has great potential for nematode diagnostics in the laboratory having minimal available equipment.

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Recombinase Polymerase Amplification assays for detection of the major tropical root-knot nematodes

Cited by 3 publications

References 11 publications

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil

Visualized Detection of Tobacco Anthracnose by a Recombinase Polymerase Amplification–Lateral Flow Dipstick Assay

Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia

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