Simple detection of hepatitis b virus in using loop-mediated isothermal amplification method

Abstract

Introduction: US Military and civilian personnel regularly deploy to regions that are endemic for the Hepatitis B virus (HBV), including the Western Pacific, Africa, Eastern Mediterranean, Southeast Asia, and Europe. When patients have life-threatening injuries that require any blood component that is not immediately available, they are typically transfused with locally collected fresh whole blood from a walking blood bank. Currently, there is no simple and easy method for sensitively screening fresh blood in deployed theaters of conflict. Materials and methods: In order to fill the gap, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of HBV in blood products. The primers were designed to target the gene of the pre-Surface/Surface antigen region of HBV. The amplification reaction mixture was incubated at 60°C for 60 min. The amplicon can be detected by a handheld fluorescence tube scanner or an immune-chromatography test strip. Results: We were able to detect down to 10 copies of viral DNA by LAMP reaction for HBV DNA extracted from HBV-positive plasma. We also identified the optimal heat treatment condition (125°C for 10 min) for plasma specimens without requiring DNA extraction for the LAMP assay. The sensitivity of the assay was evaluated with polymerase chain reaction (PCR) confirmed HBV-positive samples. Using LAMP, we detected HBV in 107 out of 127 (84%) samples. Conclusion: This LAMP assay has the potential to be used in resource-limited settings to improve the safety of locally collected blood in endemic regions.