A simple PCR method for the detection of pathogenic spiroplasmas in crustaceans and environmental samples

A simple PCR method that uses a Chelex-100 extraction has been developed to detect the 16S rRNA gene of pathogenic spiroplasmas in commercially important crustaceans. The method detected spiroplasmas in the Chinese mitten crab Eriocheir sinensis, and the crayfish Procambarus clarki, as well as from environmental samples from host habitats. Compared with the Tissues/Cell Genomic DNA Isolation Kit method, the Chelex-100 method was more sensitive to the detection of spiroplasmas from all sources sampled, with results being obtained within 4 h. The detection limit was 1 CCU/mL. This is the first time that the Chelex-100 extraction method has been used to detect spiroplasmas from cultured crustaceans. It is a simple, rapid, sensitive and low-cost method and can be applied to broad, field-based surveys.