Multiplex qPCR Assay for HIV-1 Proviral DNA Detection and Subtype Characterization: Exploiting Quenching of Multiple Fluorophores with a Single Quencher Operating in Trans

Accurate detection and quantification of HIV-1 proviral DNA are critical for effective patient monitoring and therapeutic decision-making. In this study, we developed a multiplexed quantitative PCR (qPCR) assay designed to detect HIV-1 proviral DNA, determine viral subtype, specifically identifying the predominant subtype C and validate assay performance using an internal control. Gene-specific primers were engineered by appending an 8-base biotag followed by a common 18-base sequence at the 5′ end, enabling the simultaneous amplification of multiple target sequences. Fluorescent probes labeled with FAM, SUN/VIC, and Cy5 were employed for detection, and a novel strategy involving quenching of labeled probes in trans was implemented to enhance assay flexibility and cost-effectiveness compared to conventional cis-quenched probes. The assay was initially optimized using synthetic linear double-stranded DNA templates representing the HIV-1 gag region, while externally added human chromosomal DNA served as a control for PCR inhibition. Validation was performed on a panel of 11 clinical samples previously analyzed for drug resistance mutations. Results indicated robust amplification of HIV-1 proviral DNA, accurate subtype determination, and reliable internal control performance, with profiles closely matching those obtained by gold standard sequencing-based assays. One sample exhibited PCR inhibition, underscoring the need for internal control monitoring. Overall, the multiplexed qPCR assay provides a sensitive, specific, and efficient tool for comprehensive HIV-1 reservoir quantification and molecular epidemiological studies, potentially informing improved clinical management and personalized treatment strategies. Furthermore, this novel methodology significantly reduces reagent costs and processing time while maintaining high sensitivity, making it ideal for routine clinical and research applications.