Molecular detection of Fusarium graminearum causing head blight of wheat by loop mediated isothermal amplification (LAMP) assay

Abstract

A rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed for the detection of Fusarium graminearum, predominant Fusarium spp. for causing head blight of wheat in many wheat growing countries including India. The four LAMP primers were designed from partial sequence of Fusarium graminearum chromosome 1, complete genome (accession No. HG970332.2 using region (GenBank Accession No. AY937106.1), unique to F. graminearum. The reaction temperature and ratio of outer/inner primers and optimal Mg2 + concentration for reaction efficiency were standardized. Colorimetric detection of LAMP assay showed the presence of blue colour in F. graminearum, while violet color was observed in negative reaction after adding hydroxyl napthol blue (HNB) dye. Likewise, adding 0.6 mg of ethidium bromide/ml to LAMP products showed full brightness only in positive reactions. Further, a ladder like banding pattern confirms the positive reactions on agarose gel electrophoresis whereas no patterns were observed in negative reactions. Upon amplification in the LAMP assay, only three samples out of eight tested positive by showing blue color and fluorescent signal. The assay has been optimized for speed, sensitivity, and specificity and it can detect less than 100 fg of target DNA template per reaction within 60 min. This could detect latent infection in both wheat inflorescence and seeds. Our results indicated that LAMP offers an excellent method to detect F. graminearum and it can be used as a point of care device for seed quarantine of F. graminearum and providing authentic information for disease management.