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Techniques for the evaluation of nucleic acid amplification technology performance with specimens containing interfering substances: efficacy of boom methodology for extraction of HIV-1 RNA.
Witt DJ; Kemper M; Organon Teknika Corporation, Durham, NC 27712, USA.;
November 30, 1999
J Virol Methods. 1999 Apr;79(1):97-111. Unique Identifier : AIDSLINE

Accurate HIV-1 RNA quantitation with nucleic acid amplification assays (NAAA) is partly dependent on overall assay design to ensure proper and reproducible functioning in the presence of endogenous interfering substances present in a clinical specimen, or exogenous interfering substances introduced as a result of specimen collection or handling. This study tested various methods of evaluating interfering substances that could potentially affect the outcome of HIV-1 RNA amplification in a NAAA. Clinical specimens from HIV-1 seronegative subjects containing various endogenous interferents were evaluated with and without an HIV-1 RNA spike to assess recovery and specificity, respectively, with a non-PCR NAAA (NASBA HIV-1 RNA QT, Organon Teknika) that incorporates Boom methodology for nucleic acid extraction. Additional specimens were prepared to simulate various circumstances that might occur during specimen preparation to result in the introduction of exogenous interferents. A retrovirus reverse transcriptase inhibitor, zidovudine (AZT), was added to plasma specimens prior to testing. NAAA results obtained with 127 total clinical specimens, 10 bacterially contaminated specimens, 5 platelet enriched specimens, 5 AZT specimens, and 30 anticoagulated specimens were consistent with the expected outcomes in the presence and absence of the HIV-1 RNA spike, giving an assay specificity of 100%. The spiked HIV-1 RNA copies in the clinical specimens reported by the assay were 99% of the copies reported for a positive index control (normal plasma plus HIV-1 RNA spike). Compared to the amplification levels of the three internal assay calibrators obtained for normal plasma controls, no differences in the amplification levels of the calibrators for each type of specimen were observed. This result indicated that the interferents examined did not affect adversely assay function. Addition of known PCR interferents (hemoglobin and heparin) and AZT to isolated HIV-1 RNA resulted in a substantial reduction of amplification and invalid results, whereas no inhibition was observed when these interferents were added to the test system prior to isolation; these results directly demonstrate the efficient removal of such interferents during the NASBA HIV-1 RNA QT isolation procedure. The several approaches to investigate interference described in this study may be utilized for the evaluation of other assays using nucleic acid amplification technology.

JOURNAL ARTICLE Anti-HIV Agents/PHARMACOLOGY Anticoagulants Bacteria/ISOLATION & PURIF Evaluation Studies Gene Amplification/*METHODS Human HIV Infections/*VIROLOGY HIV Seronegativity HIV-1/GENETICS/*ISOLATION & PURIF/PHYSIOLOGY RNA, Viral/*ISOLATION & PURIF Sensitivity and Specificity Specimen Handling Viral Load