title: Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Agri-environmental Division, Aichi Agricultural Research Center, Nagakute, Aichi, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Fukuta
authors.forename: R
authors.initials: R
authors.lastname: Takahashi
authors.forename: S
authors.initials: S
authors.lastname: Kuroyanagi
authors.forename: Y
authors.initials: Y
authors.lastname: Ishiguro
authors.forename: N
authors.initials: N
authors.lastname: Miyake
authors.forename: H
authors.initials: H
authors.lastname: Nagai
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: T
authors.initials: T
authors.lastname: Tsuji
authors.forename: F
authors.initials: F
authors.lastname: Hashizume
authors.forename: H
authors.initials: H
authors.lastname: Watanabe
authors.forename: K
authors.initials: K
authors.lastname: Kageyama
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D057230:Limit of Detection; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010838:Phytophthora / Q000235:genetics; D011775:Pythium / Q000235:genetics*; D015203:Reproducibility of Results; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer
keywords: Pythium myriotylum; annealing curve; diagnosis; loop-mediated isothermal amplification