Development And Evaluation Of Recombinase-aided Amplification Assays For Detection Of Hepatitis B Virus

Objectives Hepatitis B virus(HBV)is a widespread blood-borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma,particularly in southeast Asian and African countries where HBV is highly endemic and the budget and resources are limited.In this study,using heat-treated DNA,we developed two field applicable detection assays for HBV based on recombinase-aided amplification(RAA).One was an internal controlled duplex RAA assay using a portable real-time fluorescence detection device,another was an instrument-free visual observation assay using lateral flow dipsticks.Methods According to the conserved sequence of hepatitis B virus,the primers and probes were designed according to the principle,and the duplex real-time RAA assay and LFD-RAA assay were developed respectively,using heat-treated DNA.HBV-positive quantitative standard were tested by duplex real-time RAA assay and RAA-LFD assay for determining the sensitivity,respectively.The specificity was assessed by testing 15 serum samples of healthy donors,five EBV,and five CMV-positive serum samples.A total of 157 serum samples with HBs Ag-positive from Tangshan Gongren Hospital of Hebei were detected by two assays,respectively.In parallel,the same samples were quantified by a commercial q-PCR kit.The results of two assays were analyzed for Kappa and Mc Nemar's tests.Results Using heat-treated DNA,duplex real-time RAA assay and RAA-LFD assay can detect a standard with a minimum viral load of 10 IU/m L.Compared with commercial qPCR kit as a reference,our results demonstrated that the two assays obtained 97.18% and 95.77% of sensitivity,respectively,and the specificity was 100%,by testing a total of 157 serum samples with HBs Ag positive.Conclusions In this study,using heat-treated DNA,we developed two detection assays for HBV.We conclude that the advantages of rapidity,simplicity,portability,and visualization of proposed two assays make them great potentials in point-of-care testing of HBV infection by untrained people in resource-limited situations.Figure 8;Table 5;Reference 47...