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Equivalent amplification of intrinsically variable nucleic acid sequences by multiple-primer-induced overlapping amplification assay: Applications for universal detection and quantitation

Nature Medicine volume 5, pages 1081–1085 (1999)Cite this article

Molecular methods are important in the diagnosis of viral infections and molecular measurements of viral load have been used successfully for evaluating the prognosis of viral infections and for monitoring the efficacy of antiviral therapies1,2,3,4,5,6. So far, HIV viral load has been measured routinely with three commercially available molecular methods. The first method (Monitor, HIV-1 1.5) is based on the use of a reverse transcription (RT)-linked PCR amplification technique calibrated by an internal competitive standard made of a single concentration of an RNA transcript generated in vitro from mutant viral cDNA (ref. 7). The second (Nuclisens, HIV-1 QT) is based on the use of a nucleic acid sequence-based amplification (NASBA) technique that is calibrated by internal competitive standards consisting of three distinct RNA transcripts8, and the third (Quantiplex, HIV RNA 3.0) uses a branched DNA (bDNA) hybridization signal amplification system that is calibrated by external standards consisting of five known concentrations of an RNA transcript9. Both the PCR- and NASBA-based methods consist of amplifying specific nucleic acid sequence with a single pair of oligonucleotide primers. These tests that use a single pair of primers are restricted to limited viral genotypes and, given the known broad variations in viral genomes, are often associated with false negatives or underestimation of the viral load in clinical practice10,11,12,13,14,15. Although the bDNA technique is less genotype-restricted than those using PCR and NASBA (refs. 14–16), its application has been limited by a relatively low detection threshold of the hybridization signal amplification system compared with that of nucleic acid amplification systems12,16,17,18.

To address the problem of viral-genotype restriction observed with the conventional nucleic acid amplification methods, we developed a multi-target strategy that consists of the simultaneous amplification of multiple regions of specific HIV sequence19,20. However, this multi-target strategy still suffered underestimation because of the possible failure in the priming of intrinsically variable target sequences.

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Figure 1
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Figure 4: Relationship between plasma HIV-1 RNA detected and quantified by MUPROVAMA and Monitor (n = 221) (a), by MUPROVAMA and Nuclisens ( n = 199) (b) or by MUPROVAMA and Quantiplex (n = 215) (c).

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Acknowledgements

This work was partly supported by the Association pour la Recherche, l'Etude et le Traitement des Maladies du Sang (AREMAS).

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  1. Laboratoire d'Oncologie et Virologie Moléculaires, Hôpital Laennec, Faculté de Médecine Necker, Université René Descartes—Paris V, Paris, 75007, France

    Wei Lu, Li Cao & Jean-Marie Andrieu

  2. GeneQuant, Paris, 75015, France

    Likia Ty & Marine Arlie

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Correspondence to Wei Lu.

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Lu, W., Cao, L., Ty, L. et al. Equivalent amplification of intrinsically variable nucleic acid sequences by multiple-primer-induced overlapping amplification assay: Applications for universal detection and quantitation. Nat Med 5, 1081–1085 (1999). https://doi.org/10.1038/12520

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