Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection and Analysis of the Root-Knot Nematode Meloidogyne hapla in Soil

Abstract

Soil analysis is crucial for estimating the risk of crop damage by the root-knot nematode Meloidogyne hapla. Here, we developed an analysis assay based on Loop-mediated Isothermal Amplification (LAMP). The LAMP primers were verified for specificity against 10 different nematode species. A manual soil DNA extraction, referred to as SKMM, was developed and compared with a FastDNA kit followed by DNA purification. DNA was extracted with both methods from artificially inoculated soils as well as from naturally infested soil collected from farm fields. The primers exclusively amplified DNA from M. hapla with both colorimetric and real-time LAMP. The detection limit was 193 gene copies and 0.0016 juveniles (12 pg µL−1) per reaction. DNA concentrations and purity (A260/A230) were significantly higher using the SKMM procedure compared with the kit. From the field samples collected in 2019, DNA was amplified from 16% of samples extracted with SKMM and from 11% of samples using the kit. Occurrence of M. hapla DNA was confirmed in soil samples from two out of six field soils in 2020 using both real-time LAMP and qPCR. In conclusion, the developed real-time LAMP is a fast and specific assay for detection and quantification of M. hapla DNA in soil.

Diagnose Molecular De Espécies De Meloidogyne Por Meio Da Amplificação Isotérmica De Dna Mediada Por Loop (Lamp)

“…As quantidades dos demais componentes de um protocolo LAMP sofrem poucas variações, sendo adicionadas por reação oito unidades da enzima Bst DNA polimerase; 1 a 2 µL de DNA e tampão na concentração recomendada pelo fabricante, que geralmente é de 1X, ou seja, 2,5 µL do tampão a 10X por reação. Alguns kits disponíveis no mercado possuem master mix com tampão, dNTP's, Bst DNA polimerase, indicador colorimétrico e basta adicionar apenas a amostra de DNA, primers e água (WALIULLAH et al, 2020;OMER et al, 2022).…”

Ciências Agrárias: limites e potencialidades em pesquisa - Volume 2

“…Temperatura e tempo de reação usados em protocolos LAMP para a identificação de espécies de Meloidogyne. WALIULLAH et al, 2020;OMER et al, 2022).…”

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

“…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.…”