Development of real-time fluorescent enzymatic recombinase amplification method for severe fever with thrombocytopenia syndrome virus detection
Objective To establish a RT-ERA method for detection of severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Taking the conserved sequence of NSs gene of SFTSV as the target,the fluorescent probe and the corresponding primer were designed and synthesized according to the RT-ERA principle.The standard plasmid was constructed with pUC18 as the vector and transcripted in vitro as a positive control.The optimal probe primer combinations was screened after the gradient dilution of positive control product.The positive control product with different copy numbers were used to evaluate the sensitivity of RT-ERA.Then,the RNA of novel bunyavirus,novel coronavirus,forest encephalitis virus,dengue virus,chikungunya virus and Zika virus were used as templates to evaluate the specificity of the method.Finally,26 clinical samples were used to test the consistency with RT-qPCR.Results At 42 ℃,the fluorescence RT-ERA method could detect the minimum sensitivity of SFTSV RNA was 100 copies/µl within 40 minutes.When the SFTSV nucleic acid amplification was positive,the test results of other viruses were negative.A test of 26 serum samples of suspected severe fever with thrombocytopenia syndrome patients revealed that the sensitivity and specificity of RT-ERA were about 88.9％and 100.0％,respectively.The kappa value was 92.3％.Conclusion Considering the rapid,specific and sensitive advantages of RT-ERA,this method has certain application prospects in the detection of SFTSV.
万方数据
维普
