The qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici

Wheat loose smut caused by Ustilago tritici a seed-borne disease, is difficult to control due to the expansion of wheat planting area and difficulty of pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays were used to rapidly amplify the DNA of U. tritici. Five pairs primers for qPCR and two series primers for LAMP were designed. Firstly, the specify of primers were carried out by using the DNAs of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Then the amplification systems are optimized. Finally, the sensitivity of qPCR and LAMP assays were quantified. The results show that using the primers pairs Y430F/R, Y307F/R, Y755F/R and Y139F/R for qPCR, primers L-139 and L-988 for LAMP assay could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay is 10 pg μL^-1 of genomic DNA, the detection limit of LAMP assay is 100 fg μL ^-1 . We successfully performed qPCR and LAMP assays on two wheat loose smut wheat samples, and confirmed sequenced U. tritici infection by subsequently sequencing. This paper established two methods for U. tritici detection, which could be used for wheat loose smut diagnose in lab and field.