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

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulatin g recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quant ify all HIV-1 isolates reliably are in demand. We have developed a fast, re al-time monitored RNA assay based on an isothermal nucleic acid sequence-ba sed amplification technology that amplifies a part of the long terminal rep eat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monit ored in a fluorimeter with a thermostat. The lower le c el of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of q uantification was 100 copies of HIV-1 RNA,vith a dynamic range of linear qu antification between 10(2) and 10(7) RNA molecules. All HIV-1 groups, subty pes, and CRFs could be detected and quantified with equal efficiency, inclu ding the group N isolate YBF30 and the group O isolate ANT70, To test the c linical utility of the assay, a series of 62 serum samples containing virus es 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 commer cially available assay. This comparison showed that the quantification resu lts correlated highly (R-2 = 0.735) for those subtypes that could be well q uantified by both assays (subtypes B, C, D, and Fl, whereas improved quanti fication was obtained for subtypes A and G and CRFs AE and Ac, A retrospect ive study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group hi, who were treated with highly active an tiretroviral therapy, revealed that the assay was well suited to the monito ring of therapy effects.. In conclusion, the newly developed real-time moni tored HIV-I assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all sub types and groups of HIV-1.