Simple Detection of Hepatitis B Virus in Using Loop-Mediated Isothermal Amplification Method

Abstract

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.