The rtLC DBS assay described herein is a one-step, walk away technique. Automation of this assay provides potential for high throughput with very small sample volumes, which makes the assay suitable for use in infants and children from whom one often has access to only small blood volumes. It is an assay system which will be cost-effective and easily adaptable to limited-resource settings, where the majority of new HIV-1 infections are seen today. The detection of PCR products by SYBR Green ensures good sensitivity. SYBR Green is relatively inexpensive compared to probe-based detection, and in general, SYBR Green detection is one cycle threshold or so more sensitive than probe-based assays.