Sensitive and rapid detection of freshwater crustacean Spiroplasmas by ISRs-sequence-targeted species-specific primers

For the rapid and sensitive identification of the Spiroplasmas in the Chinese mitten crab Eriocheir sinensis, a species-specific PCR primer-pair was designed according to Spiroplasma sp. strain CRAB-specific segments of the previously sequenced 16S–23S rRNA intergenic spacer region (ISR). The PCR-mediated assay allowed identification of Spiroplasmas from different aquatic crustacean hosts, E. sinensis and red swamp crayfish Procambarus clarkia, in different geographical areas in China. No cross-reaction could be observed with other three available terrestrial plants/insects Spiroplasma strains (Spiroplasma sp. CH-1, Spiroplasma sp. CR-1 and Spiroplasma sp. M10). The sensitivity of detection of the Spiroplasma sp. strain CRAB could get as little as 100 fg of DNA template. The presented PCR method is useful for sensitive and specific detection of Spiroplasma from freshwater cultured crustaceans in the trade of aquatic products.