Colorimetric polymerase spiral reaction (PSR) assay for the rapid detection of hepatitis B virus

Abstract Hepatitis B virus (HBV) infection remains a significant global health concern, particularly in low-resource settings with limited access to sophisticated diagnostic tools. Conventional molecular diagnostic assays, such as qPCR, are often restricted to centralized laboratories in urban settings due to their high cost and the need for skilled personnel. This limits its use in lower levels of the healthcare system, highlighting the need for affordable point-of-care (PoC) assays. In this study, we have developed a visual detection method for HBV using a polymerase spiral reaction (PSR) incorporated with hydroxy naphthol blue (HNB) dye. A set of primers was designed targeting a specific region of the S gene of HBV, and the reaction runs in an isothermal condition. The assay conditions were optimized, and the optimal reaction temperature and time were 63 °C and 60 min, respectively. This developed HBV-PSR assay showed a detection limit of 5.04 copies, reaching 100% sensitivity and 100% specificity in clinical evaluation ( n = 85), and colorimetric visualization in 60 min by naked eye. The specificity test revealed no cross-reactivity with other hepatitis-causing viruses and the tested non-HBV pathogens. The assay was tested with 45 HBV-positive clinical samples, which were initially screened by a serological test, and showed a good concordance rate and diagnostic reliability. The HBV-PSR assay designed in this study could potentially be used as a point-of-care diagnostic tool for HBV detection, aiming at early surveillance and detection, focusing on healthcare in poor-resource settings. The combination of isothermal amplification with dye-based visual detection eliminated the need for complex instruments, enabling PoC detection. Key points • A rapid and sensitive PSR assay was developed for HBV detection, with results obtained within 1 h . • HNB dye enabled naked eye visual detection without the need for complex equipment . • The assay detected as few as 5.04 DNA copies and demonstrated high diagnostic performance, outperforming conventional PCR in the evaluated clinical samples .