Abstract:

Objective To establish a novel and rapid method to detect Schistosoma japonicum specific gene fragment using a real-time recombinase aided isothermal amplification (RAA) combined with a fluorescent probe. Methods S. japonicum G28（SjG28） was selected as the target gene to be detected, and the primers and fluorescent probes were designed accordingly. A real-time fluorescent RAA using RAA combined with fluorescent probe was established to detect SjG28. The sensitivity and specificity of the real-time fluorescent RAA was determined and optimized by using gradient dilutions of SjG28 recombinant plasmid as template and the genomic DNAs from other helminths of S. mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides and Clonorchis sinensis as controls. Then this method was applied to detect SjG28 in genomic DNA extracted from snails and feces of rabbits infected with S. japonicum. Results As low as 10 copies of SjG28 plasmid DNA could be detected by the developed real-time fluorescent RAA within 10 min. This method was able to detect SjG28 in the fecal samples from rabbit infected with different dose of cercarias (20, 200 and 500) within 15 min and snail pool (50) mixed with 1, 2, 3, 4, 5 and 10 cercaria-positive snails within 10 min. There was no product amplified from genomic DNAs extracted from other helminths: S. mansoni, E. granulosus, A. duodenale, A. lumbricoides and C. sinensis. Conclusion A novel and rapid florescent real-time fluorescent RAA method was established to detect S. japonicum with high sensitivity and specificity.

Key words: Schistosoma japonicum, Gene detection, Isothermal amplification of nucleic acid, Fluorescent probe, Recombinase