Abstract
BackgroundHuman Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India.ResultsLinear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p < 0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p = 0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000–25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians.ConclusionThe HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.
Highlights
Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients
We report a real time RT-PCR assay optimized for HIV-1 C subtype that uses SYBR Green chemistry for quantitative detection of HIV-1 viral RNA in human plasma
In this study we described a SYBR Green chemistry based sensitive real time PCR assay for quantitative detection of HIV-1 viral load in human plasma samples
Summary
Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is an assay that is economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. The problem of sub-optimal performance by TaqMan chemistry based commercial assay is mostly encountered in countries where non B subtype of HIV-1 is prevalent because these assays are widely validated in USA and Europe where HIV-1 subtype B dominates [3,5]. In addition to the TaqMan chemistry, SYBR Green chemistry based real time PCR is widely used for HIV-1 viral RNA quantification assays
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