RESUMEN
UNLABELLED: This study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 87·0°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Pythium/genética , Cartilla de ADN/genética , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Límite de Detección , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Phytophthora/genética , Reproducibilidad de los Resultados , Microbiología del SueloRESUMEN
To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5'-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.
