Molecular Diagnosis of Viral Hepatitis B Infection v1

Abstract

With over 400 million HBV infections, viral hepatitis B remains a global public health concern. Diagnosis is primarily based on an immunological assay approach, which utilizes the Hepatitis B surface antigen in detection amongst other markers. This method, however, has several limitations which include the inability to detect mutation in the viral genome resulting in diagnostic escape and low antigen titers in study samples. Using an alternative approach, which is the molecular diagnostic technique such as real-time polymerase chain reaction (RT-PCR) would circumvent the limitations of immune detection. This protocol, thus, provides a step-by-step process of HBV diagnosis using RT-PCR which is a sensitive tool for diagnosis. The steps involved include sample collection and preparation, nuclei acid isolation, HBV detection and quantification using RT-PCR as well as the interpretation of results. This protocol combines the high nuclei acid yield from isolation using Zymo Quick DNA Mini-prep Kit and Bosphore HBV Quantitative Kit for amplification of HBV DNA. The Bosphore HBV Quantitative Kit has a low detection limit of 1×101 IU/ml and with a turn-around time of less than 4 hours when combined with Zymo Quick DNA Mini-prep Kit. The nucleic acid isolated in this protocol can be amplified using the Bosphore HBV Quantitative kit on several thermocyclers, which makes the protocol robust, cost-efficient, and cost-effective in resource-scarce areas.