Development of a loop-mediated isothermal amplification method for the rapid detection of Pythium ultimum

Abstract

Pythium ultimum is an important soil-borne pathogen that causes damping-off and root rot in a large number of plant species. We developed a loop-mediated isothermal amplification (LAMP) method for the rapid and accurate detection of P. ultimum. A target gene encoding a spore wall protein was identified from the P. ultimum genome using a comparative genomics approach. The LAMP assay efficiently amplified the target DNA at 64 °C for 60 min, and its specificity and sensitivity were evaluated. Of the pathogen isolates tested, positive results were obtained only with P. ultimum isolates. The detection limit of the LAMP method was 1 pg μL−1 DNA, which is 1000 times more sensitive than conventional PCR. Moreover, this method was successfully used to detect P. ultimum DNA extracted from infected plant tissues, and the detection limit for P. ultimum in plants was nearly the same as for in vitro samples. Consequently, our LAMP assay enables the rapid, specific, and sensitive detection of P. ultimum DNA from pure cultures, as well as from infected plant tissues.