Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

Abstract

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was >or=99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 - specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both >or=99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.

FIG. 1.

Cycle of transcription-mediated amplification. TMA includes the following five main activities. (i) A promoter-primer binds to the RNA target and is extended via the DNA polymerase activity of the reverse transcriptase (RT) (steps 1 and 2). The product of this reaction is an RNA/DNA hybrid duplex, with the new DNA strand being complementary to the virion RNA target (step 3). (ii) RNase H activity of the reverse transcriptase specifically digests the RNA strand of the RNA/DNA hybrid, leaving only the cDNA (step 4). (iii) Following the RNA degradation, the non-T7 primer (i.e., the primer without the T7 promoter) base pairs to its complementary sequence on the newly created DNA strand, and the reverse transcriptase catalyzes the synthesis of another new DNA strand using the cDNA as a template (step 5). The product of this reaction is a DNA intermediate that contains the bacteriophage T7 RNA polymerase promoter sequence in double-stranded form (step 6). (iv) The bacteriophage T7 RNA polymerase now recognizes the promoter sequence on the DNA intermediate and transcribes multiple copies of the RNA amplicon (steps 7 and 8). The RNA amplicon strands are opposite in polarity from the original RNA target and contain a region complementary to the probe used for amplicon detection. (v) The RNA strands created in iv are able to base pair to the non-T7 primer (step 9). Subsequently, the reverse transcriptase catalyzes the formation of a cDNA strand by extending the 3′ end of the non-T7 primer, and again an RNA/DNA hybrid duplex results (step 10) as in i, repeating the cycle.

FIG. 2.

Distribution of S/CO ratios of normal blood bank specimens and calibrators tested with the TMA HIV-1/HCV assay. Approximately 1,500 specimens from healthy donors were analyzed in the HIV-1/HCV assay, and their resulting internal control (IC) and analyte S/CO ratios are plotted as black and gray bars, respectively (A). The cutoff values used for the S/CO calculation were obtained from the formulas listed in Table 1. (B) Distribution of analyte S/CO ratios for the calibrators included in the specificity analysis assay runs. Bars represent the number of reactions at each S/CO, with black, white, and hatched bars representative of the negative calibrator, HIV-1 positive calibrator, and HCV positive calibrator, respectively.

FIG. 3.

ROC plots of the specificity and sensitivity of the TMA HIV-1/HCV assay, the HIV-1 discriminatory assay, and the HCV discriminatory assay. Specificity and sensitivity data were assessed for each assay by ROC analysis as described in Materials and Methods. The results represent the plots of the TMA HIV-1/HCV assay detection of HIV-1 (A), the TMA HIV-1/HCV assay detection of HCV (B), the HIV-1 discriminatory assay (C), and the HCV discriminatory assay (D). The scale of the y axis is limited to better show the sensitivity and specificity limits.