ABSTRACT
Objective To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni. Methods The 121 bp highly-repeated sequence of S. mansoni was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum, S. haematobium, Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity. Results A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum, S. haematobium, A. duodenale and C. sinensis. Conclusions A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni.
ABSTRACT
Objective To establish a novel method for the detection of Schistosoma japonicum specific gene fragments by re-combinase aided isothermal amplification(RAA).Methods The gene fragment SjG28 of S.japonicum was selected as the tar-get 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.Results 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 with-in 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.Conclusion A novel method for the detection of S.japonicum specific gene fragments by re-combinase 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.