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Journal of Clinical Microbiology, December 2001, p. 4302-4308, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4302-4308.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

TaqMan 5'-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

C. Drosten,dagger E. Seifried, and W. K. Roth*

Institute of Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service, Frankfurt am Main, Germany

Received 21 March 2001/Returned for modification 19 July 2001/Accepted 14 September 2001

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >= 95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.

* Corresponding author. Mailing address: Institut für Transfusionsmedizin und Immunhämatologie, Blutspendedienst des DRK Hessen, Sandhofstr. 1, D-60528 Frankfurt am Main, Germany. Phone: 0049-69-6782-251. Fax: 0049-69-6782-256. E-mail: wroth{at}bsdhessen.de.

dagger Present address: Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany.

Journal of Clinical Microbiology, December 2001, p. 4302-4308, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4302-4308.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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