Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood

Abstract

Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) based methods over serological tests and PCR for rapid detection of microbial pathogens. Here, a rapid LAMP assay was developed to detect the hepatitis B virus (HBV) from DNA, and particularly, blood samples from infected patients using a commercially available master mix and portable real-time fluorometer. The final optimized fluorescence-based LAMP assay provided significant amplification time of less than 15 minutes compared with over 1 hour for PCR and an opened tube LAMP system described previously. Results indicated that fluorescence-based LAMP assay was more sensitive than PCR as a rapid, sensitive, efficient, and highly reliable approach for rapid detection of HBV.

Keywords: Biochemistry; Bioengineering; Evidence-based medicine; Infectious disease; Medicine; Microbiology; Pathology; Virology.

Fig. 1

Gel electrophoresis of PCR and LAMP assays with different templates. (A) PCR reactions with HBV-DNA (lane 1), heat-treated plasma specimen (lane 2), heat-treated blood sample (lane 3). (B) LAMP assays with HBV-DNA (lane 1), heat-treated plasma specimen (lane 2), heat-treated blood sample (lane 3).

Fig. 2

Detection of Hepatitis B Virus (HBV) from heat-treated blood samples using the Genie III system (OptiGene, UK). (A) Electrophoresis result of LAMP to blood heat-treatment. (B) Amplification plots of the LAMP assays from blood heat-treatment. (C) Electrophoresis results of HBV-DNA from blood samples using PCR method with F3 and B3 primers. Lane M = 1 kp marker; Lanes 1–7 = HCM.F.37, HCM.F.32.1, HCM.F.62, HCM.M.41, HCM.F.29, HCM.F.32.2, HCM.F.28 DNA-HBV samples; Lane 8 = Negative LAMP assay.