qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici
Loose smut of wheat, caused by the basidiomycete fungus Ustilago tritici, is a seed-borne disease that is challenging to control due to the vast areas of wheat cultivation and the difficulty in detecting the pathogen. This study introduces real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid detection of U. tritici DNA. We designed five pairs of primers for qPCR and two sets of primers for LAMP. The specificity of these primers was tested using genomic DNA from U. tritici and several other fungi, including Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani. The amplification systems were optimized, and the sensitivity of both qPCR and LAMP assays was evaluated.
Our results indicate that primers Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR, as well as primers L-139 and L-988 for LAMP, are effective for detecting U. tritici. The sensitivity tests revealed that the qPCR assay can detect as little as 10 pg/μL of genomic DNA, while the LAMP assay can detect as little as 100 fg/μL. Both qPCR and LAMP assays were successfully applied to wheat samples infected with loose smut. This study establishes two reliable methods for detecting U. tritici, which can be utilized for diagnosing wheat loose smut both in the laboratory E7766 and in the field.