Establishment of a recombinase-aided isothermal amplification technique to detect Schistosoma japonicum specific gene fragments

Abstract

To establish a novel method for the detection of Schistosoma japonicum specific gene fragments by recombinase aided isothermal amplification (RAA). The gene fragment SjG28 of S. japonicum was selected as the target gene fragment to be detected, and the primers were designed according to the mechanism of RAA reaction. The reaction of isothermal amplification of S. japonicum was established and optimized. Then this method was applied to amplify and detect the specific gene fragment in the gradient diluent SjG28-recombiant plasmids and different concentrations of S. japonicum genomic DNA to estimate the sensitivity of this method. The samples were also detected by polymerase chain reaction (PCR) in parallel as control. This method was applied to detect the genomic DNA of S. mansoni, Ascaris lumbricoides, and Ancylostoma duodenale to evaluate the specificity. The specific gene fragment was amplified from genomic DNA of adult worms and eggs of S. japonicum by recombinase aided isothermal amplification reaction established in this study. The reaction can be completed within 30 minutes and the minimum detectable template was 20 copies of plasmids or 0.5 ng of genomic DNA per microliter. Other parasites'genomic DNAs, such as S. mansoni, A. lumbricoides, An. duodenale and healthy human blood genomic DNA were not able to be detected by this method. A novel method for the detection of S. japonicum specific gene fragments by recombinase aided isothermal amplification is established in this study, which can be carried out conveniently and rapidly with a considerable sensitivity and specificity, showing the prospect for application in the diagnosis of schistosomiasis japonica.