Table 1.

Selected technologies used in quantitative HIV DNA real time PCR assays.

Assay	Normalisation	Unique Features and Advantages/Disadvantages	PCR Template Loading Quantity	LOD	Reference
Whole blood ANRS LTR real time PCR Method	HIV-DNA copies/µg converted to copies/million leucocytes	HIV DNA extracted from re-suspended whole blood cell pellets No separation of cells Its quicker and cost serving Normalisation using cell count/cytometry is not accurate	1 µg DNA, equivalent to 150,000 cells	1 Copy	[34]
LTR ANRS LTR real time PCR Method	Copies/million/PBMC	First assay for estimating HIV reservoir size to be evaluated for inter-laboratory reproducibility. The assay available as a commercial	1 µg DNA, equivalent to 150,000 cells	1 Copy	[34]
SYBR Green gag HIV DNA PCR	Copies/hundred thousand cells	Uses SYBR green fluorescence and amplifies a 142-gag fragment The assay is not sensitive especially in suppressed patients	300 ng, equivalent to 1,000,000 cells	50 Copies	[27]
Cross-Clade Ultrasensitive nested PCR	CD3 gene copy cell equivalents	Assay is performed on cell lysate, hence circumvents laborious nucleic acid extraction Allows quantification of HIV DNA A, B, C, D, CRF01_A/E, and CRF02_A/G HIV-1 subtypes, targeting highly conserved LTR-gag region. Cell input equivalence is accurately quantified by CD3 gene quantitative PCR. It has a disadvantage of 2 rounds of PCR	100,000 Cells	3 Copies	[91]
universal real-time PCR for group-M HIV-1 DNA	CCR5 gene copy cell equivalence	HIV-1 M-specific quantitative measures the HIV-1 Proviral DNA load in group M with a similar degree of sensitivity and accuracy across subtypes. Uses cell lysate as a template Predilution required if loading quantity is above 150,000 cells	1 µg per reaction, equivalent to 150,000 cells	1 Copy	[92]
Novel Assay for Total cell associated HIV-1 DNA	CCR5 gene copy cell equivalence	Sensitive Quantitative real time PCR for HIV-1 Cell associated DNA (CAD) targeting 3’ region of the pol gene. Assay involves enhanced nucleic extraction by ensuring adequate cell lysis through ultrasonic cell disruption	1.7 µg	3 Copies	[82]