Since in India subtype C is the predominant circulating HIV-1 clade accounting for more than 90% infections, we at Dr. Lal Pathlabs Pvt. Ltd. conceptualised development of a sensitive, specific, reproducible and affordable assay based on Real-time Reverse Transcriptase-Polymerase Chain Reaction technique (RT-PCR) for quantification of plasma HIV-1 RNA levels in patients with HIV infection. The real-time PCR technology measures accumulation of PCR product in real-time providing better quantification of the initial copy numbers with a dynamic range of detection. We designed and developed a two-tube method in which one tube is used for reverse transcription of HIV-RNA extracted from plasma/serum of HIV infected individual and the second tube is used for amplification of the transcribed cDNA. The use of Taqman probe contributes in measuring reporter dye emissions accurately with almost 100% specificity and sensitivity since it has already been reported that a single mismatch can lead to reduced amplification efficiencies resulting in decreased viral load measurements. Our assay is highly specific as all HIV-1 negative healthy donor samples failed to show any positive fluorescent signal. In addition the assay has a very high sensitivity and it can detect 30 g. eq./ml of HIV RNA. The standard curve showed linearity over eight orders of magnitude with high coefficient of correlation indicating its high sensitivity in determining viral loads over a broad range.

Uniqueness of this test resides in

• It quantitated almost all subtypes of HIV-1 including HIV-1 subtype O, particularly  ● subtype C and subtype B;