Development of colorimetric and fluorescent closed tube LAMP assay using simplified extraction for diagnosis of Meloidogyne enterolobii in root tissues

Abstract

Meloidogyne enterolobii, a guava root-knot nematode, is a highly virulent pest in tropical and subtropical regions causing galls or knots in roots of diverse plant species posing a serious threat to agriculture. Managing this nematode is challenging due to limitations in conventional identification based on isolation and microscopic classification requiring expertise and time. A colorimetric and fluorescent LAMP assay using simplified extraction method targeting rDNA-ITS region was developed to detect M. enterolobii DNA. The Men-LAMP assay exhibits simple procedure and achievable outcomes directly from root gall samples within 75 to 80 min, using a simplified Worm Lysis Buffer Plus (WLB +) extraction and the LAMP assay. The results could be interpreted using color and fluorescence without requiring post-amplification to minimize any possibility of contamination. The specificity showed no cross amplification with other plant-parasitic nematodes, a sensitivity was limited to 2.89 ng/μL. Our study proposes a sensitive, specific and time-efficient diagnostic tool for M. enterolobii infection as an alternative promising method for rapid and effective diagnosis at point-of-service to manage and control of M. enterolobii in export plants that can contribute to the degradation of trade restrictions and streamline of the international quarantine inspection process.

Keywords: Meloidogyne enterolobii; Extraction; LAMP; Parasite; Root; SYBR.

Fig. 1

Optimization of Men-LAMP assay to detect M. enterolobii DNA, (a) effect of incubation temperature, (b) effect of incubation time. Abbreviations of variable names: no template control (NTC), M. enterolobii (Me), visible light (VL), fluorescent light (FL), base pair (bp), 100 bp Plus DNA ladder (M).

Fig. 2

Specificity and sensitivity of the Men-LAMP assay, (a) LAMP detection of different plant-parasitic nematode species based on colorimetric and fluorescent closed tube LAMP assay: 1–9; M. enterolobii, 10–22; M. incognita, M. graminicola, Aphelenchus sp., Aphelenchoides sp., Pratylenchus curvicauda, Tylenchorhynchus sp. and Helicotylenchus dihystera, respectively, (b) LoD of the LAMP assay using color, fluorescence and gel electrophoresis detection (ten-fold serial dilutions; 289 μg/μL to 2.89 pg/μL), (c) LoD of real-time PCR, (d) LoD of PCR. Abbreviations of variable names: no template control (NTC), M. enterolobii (Me: 10⁰ dilution), visible light (VL), fluorescent light (FL), base pair (bp), 100 bp Plus DNA ladder (M). The gel of (a) was grouped from upper and lower parts of the same gel. Original gel is presented in Supplementary Figure S3.

Fig. 3

Effects and optimization of incubating WLB + method for extracting M. enterolobii DNA directly from root gall samples of Okra plants on Men-LAMP assay, (a) detection range of non-incubation at 1 to 10⁻² dilutions, (b) detection range of incubation at 1 to 10⁻² dilutions, (c) sensitivity of 1 to 10⁻¹ dilutions at different incubation time (15 to 90 min). Abbreviations of variable names: no template control (NTC), M. enterolobii J2 isolate (Pos), M. enterolobii extracted from root samples (Me: 10⁰ dilution), visible light (VL), fluorescent light (FL), base pair (bp), 100 bp Plus DNA ladder (M). The upper and lower gels of (c) were cropped from the same gel. Original gel is presented in Supplementary Figure S8.

Fig. 4

Comparison of sensitivity and efficiency between simplified WLB + extraction and plant material DNA extraction kit (OptiGene Lysis Buffer) using Men-LAMP assay, (a) LoDs of WLB + (left) (ten-fold serial dilutions; 179 ng/μL to 1.79 fg/μL) and OptiGene Lysis Buffer (right) (ten-fold serial dilutions; 159 ng/μL to 1.59 fg/μL), (b) detection of root-knot M. enterolobii DNA in infected root gall plants by the WLB + (left, n = 10) and OptiGene Lysis Buffer (right, n = 10). Abbreviations of variable names: no template control (NTC), M. enterolobii extracted from root samples (Me: 10⁰ dilution), visible light (VL), fluorescent light (FL), base pair (bp), 100 bp Plus DNA ladder (M).

Fig. 5

Schematic procedure of root-knot M. enterolobii detection in export plants using colorimetric and fluorescent closed tube LAMP assay based on simplified extraction method.