Loop-mediated isothermal amplification assay for sensitive and rapid detection of Globisporangium recalcitrans

Abstract

Globisporangium recalcitrans is an important phytopathogenic oomycete that causes soybean root rot. To develop a loop-mediated isothermal amplification (LAMP)-based system for rapid and specific detection of G. recalcitrans, four LAMP primers and two loop primers were designed with the Ribosomal DNA transcribed spacer 2 sequence (ITS2) as the target gene. The specificity and sensitivity of these primers were validated, and the system was also successfully applied to detect G. recalcitrans in soybean tissues after artificial inoculation and natural infection. The nucleic acid amplification reaction was performed under isothermal conditions at 63 °C for 60 min. Specificity was compared with those for 71 strains of G. recalcitrans, other Globisporangium spp. The minimum detection limit of the system was 100 fg·μL⁻¹ for detecting fungal genomic DNA. This LAMP-based system provides a technique for specific detection of G. recalcitrans and rapid diagnosis of the disease it caused.