Performance of a multiplex qualitative PCR LCx assay for detection of human immunodeficiency virus type 1 (HIV-1) group M subtypes, group O, and HIV-2

Abstract

Early detection of human immunodeficiency virus (HIV) in blood and blood products can be achieved by a sensitive nucleic acid amplification-based assay. We report on the performance of a PCR-based qualitative assay that detects both HIV type 1 (HIV-1) and HIV-2 with a sensitivity of 20 to 50 copies/ml. The assay has a specificity of 99.6% and an inhibition rate of 1.7%. One milliliter of sample is processed with a manifold system and Qiagen columns, and one-third of the extracted sample is used for PCR amplification. An internal control sequence, which is processed and amplified with each sample, monitors for amplification inhibition. Samples are reverse transcribed and are then amplified by reverse transcription-coupled PCR, after which HIV-1- and HIV-2-specific probes are hybridized to the amplified products. Following hybridization, samples are detected in the LCx instrument by microparticle enzyme immunoassay techniques. The detection system has an automated inactivation step that controls for PCR contamination. The HIV-1/2 qualitative RNA assay detects HIV-1 group M subtypes A, B, C, D, E, F, and G and group O. Testing of several HIV-1 seroconversion panels has demonstrated that the HIV-1/2 qualitative RNA assay detects HIV infection on the average of 6 days before p24 antigen can be detected and 11 days before antibodies can be detected.

FIG. 1

(A) Analytical sensitivity of the LCx HIV-1/2 qualitative RNA assay with virion panels. HIV-1 virion panels quantitated against viral quality assurance standards were diluted in negative human plasma to the indicated concentrations. One milliliter of each sample was processed as described in Materials and Methods, and one-third of each of the processed samples (0.3 ml plasma equivalent) was tested. Six replicates of each concentration were tested. Percent positive samples are indicated above the bars. An S/CO of >1 is considered a positive result. (B) Analytical sensitivity of the LCx HIV-1/2 qualitative RNA assay with transcript panels. HIV-1 and HIV-2 transcripts quantitated by hyperchromicity were tested with the indicated number of copies per reaction mixture as described in the Materials and Methods section. Six replicates of each concentration were tested. Percent positive samples are indicated above the bars. An S/CO of >1 is considered a positive result.

FIG. 2

Sensitivity of the LCx HIV-1/2 qualitative RNA assay with HIV-1 subtype A to F clones. Cloned fragments containing a region of the pol genes from HIV-1 subtypes A to F were transcribed in vitro. The transcripts were quantitated by hyperchromicity, diluted to the indicated concentrations, and tested by the HIV-1/2 qualitative RNA assay. An S/CO of >1 is considered a positive result. rxn, reaction.