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

Abstract

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
• LAMP
• Extraction
• SYBR
• Root
• Parasite