Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

Abstract

A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of species-specific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`-CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germ-free seedcanes.

Keywords: Sugarcane, Ustilago scitaminea, nested-polymerase chain reaction (PCR), molecular detection