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

Background: Hepatitis B virus (HBV) remains a major global health concern, and early detection is essential to improve clinical outcomes. Conventional diagnostic methods are often limited by cost and performance. This study aimed to establish and validate a low-cost SYBR green-based real-time PCR assay for HBV detection. Methods: A SYBR green real-time PCR assay targeting the S gene of HBV was established. DNA was extracted from plasma using a commercial nucleic acid extraction kit. Analytical specificity was assessed against clinically relevant pathogens, and Analytical sensitivity was determined using 10-fold serial dilutions of HBV DNA (10⁴–10⁰ IU/mL). Clinical validation was conducted on 112 patient samples and compared with a commercial PCR kit. Statistical agreement was evaluated using Chi-square analysis. Results: The assay showed 100% analytical specificity and a limit of detection of 10 IU/mL. Validation with 112 samples demonstrated strong concordance with the commercial kit. The in-house assay detected 45 positives and 67 negatives, compared with 47 positives and 65 negatives by the commercial assay. Only two cases were missed, resulting in a diagnostic sensitivity of 97.01% (95% CI: 89.75–99.18) and diagnostic specificity of 100% (95% CI: 89.11–100.00). The positive predictive value was 100%, and the negative predictive value was 97.01%. Agreement was highly significant (Pearson’s Chi-square = 104.03, p < 0.001). Precision testing showed an interassay coefficient of variation below 1%, indicating excellent reproducibility. Conclusions: The establishment and validation SYBR green real-time PCR assay is highly sensitive, specific, and reproducible. Its strong agreement with commercial assays and reduced cost highlight its potential as a reliable diagnostic tool as a routine laboratory method in resource-limited settings.