Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O

Abstract

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10(2) and 10(7) RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R(2) = 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.

FIG. 1

(A) Amplification plots of a tenfold serial dilution series of calibrator RNA synthesized in vitro. The amount of input RNA was 5 × 10⁷, 5 × 10⁶, 5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹, or 0 molecules. (B) Box plot depicting calculated TTP versus amount of input RNA copies from five replications of 10-fold serial dilutions of calibrator series, as described for panel A (5.0E+02 = 5.0 × 10², etc.). The box represents the interquartile range. The “whiskers” that extend from the box indicate the highest and lowest values, excluding the outliers, which are separately plotted (○). The line across each box indicates the median.

FIG. 2

Measured TTP in minutes versus log₁₀ of the dilution factor for the group O isolate ANT70 (●), the group N isolate YBF30 (■), and as a reference, the calibration curve (○).

FIG. 3

Scatter diagrams of log₁₀ RNA levels (copies/milliliter) as assessed by the Retina HIV-1 assay versus the log₁₀ RNA levels (copies/ml) as assessed by the NucliSens assay. Depicted are the results from individuals infected with HIV-1 subtypes B (n = 11), C (n = 7), D (n = 9), F (n = 1), and H (n = 1) (A) and subtypes A (n = 12) and G (n = 6) and CRFs AE (n = 5) and AG (n = 10) (B). The horizontal and vertical lines depict the lower detection level of each assay, whereas the diagonal line represents the regression line through the origin.

FIG. 4

Viral RNA levels in sera from five longitudinally followed HIV-1-infected individuals who were treated with antiretroviral therapy. Depicted are the results from both Retina HIV-1 (●) and NucliSens (○) assays. Each panel depicts the results from an individual infected with the indicated subtype of virus.