A cost-effective SYBR Green based Real-Time PCR platform for reliable detection of Hepatitis B virus: Establishment and clinical validation

Hepatitis B virus (HBV) remains a major global health challenge, and accurate, rapid, and affordable molecular diagnostics are essential for timely detection, particularly in resource-limited settings where commercial molecular assays are often inaccessible. This study aimed to establish and validate an in-house SYBR Green-based real-time PCR assay for qualitative detection of HBV DNA in serum and plasma as a reliable and cost-effective alternative to commercial assays. An in-house SYBR Green real-time PCR assay targeting the HBV S gene was developed and validated. Analytical specificity was evaluated using clinically relevant pathogens, while analytical sensitivity was determined by serial dilutions of HBV DNA. Clinical performance was assessed using 112 patient samples and compared with a commercial real-time PCR assay. The assay demonstrated 100% analytical specificity and a limit of detection of 10 IU/mL. Diagnostic sensitivity and specificity were 95.74% (95% CI: 85.75–98.83) and 100% (95% CI: 94.42–100.00), respectively, with a positive predictive value of 100% and a negative predictive value of 97.01%. Agreement with the commercial assay was significant (Pearson’s χ² = 104.03, *p* < 0.001), and inter-assay variability was < 1%. The in-house assay reduced reagent costs by approximately 78% (US$5.32 vs. US$24.69 per test) while maintaining diagnostic performance comparable to the commercial reference assay, supporting its application for routine HBV diagnosis in resource-limited settings.