Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2022

Development of point-of-care test for hepatitis B virus (HBV) infection. (#130)

Rayid K. Qasim 1 2 3 , Jack Richards 1 2 , David Delgado-Diaz 1 , Charles Narh 1 , Hanah Nguyen 1
  1. Zip diagnostics, Collingwood, VIC 3066, Australia
  2. Central Clinical School, Monash University, Melbourne, VIC 3004, Australia
  3. College Of Pharmacy, Umm Al-Qura University, Makkah, Western Province 24230, Saudi Arabia

Introduction

Hepatitis B virus (HBV) is the most prevalent blood-borne pathogen globally, infecting approximately 296 million people. HBV infects the liver and can establish a chronic infection leading to life-threatening cirrhosis and hepatocellular carcinoma (1). The development of new point-of-care tests to detect HBV viral replication would be a significant clinical tool and would contribute to reduce the public health burden of the disease. Loop mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology with potential for the detection of infectious microorganisms, including HBV (2). This technology requires six primers, and a DNA polymerase enzyme with strand displacement activity, which amplify the target gene at a constant temperature (3, 4).

Aim

The aim of this research project is to develop a point-of-care test for the rapid detection of HBV DNA from blood derived specimens, that could be deployed in resource-limited settings.

Method

Pan-genotypic primer sets were designed to amplify conserved HBV gene regions. An inclusivity testing was conducted and confirmed detection of all HBV genotypes. Optimisation of the assay components using commercially available reagents were carried out and compatibility of the assay with whole blood and plasma samples was assessed. The role of surface-active agents (detergents) in enhancing the assay performance was investigated.

Results

The assay successfully detected all major HBV genotypes (A/B/C/D/E/F/H). An extraction-free sample preparation method involving 20-fold dilution, and heating at 95°C for 5 minutes was identified to be optimal for plasma processing, yielding the fastest Tp (time to positivity). The use of detergents or surface-active agents such as digitonin did not significantly improve the performance of the assay. The optimum temperature and duration of amplification for the assay was 65oC for 60 minutes.

  1. Fact sheet: Hepatitis B [Internet]: World Health Organization (WHO); [cited 2022 21 April]. Available from: https://www.who.int/news-room/fact-sheets/detail/hepatitis-b.
  2. Chen CM, Ouyang S, Lin LY, Wu LJ, Xie TA, Chen JJ, et al. Diagnostic accuracy of LAMP assay for HBV infection. Journal of clinical laboratory analysis. 2020;34(7):e23281.
  3. Parida M, Sannarangaiah S, Dash PK, Rao P, Morita K. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Reviews in medical virology. 2008;18(6):407-21.
  4. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic acids research. 2000;28(12):e63-e.