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Journal of Clinical Microbiology, March 1998, p. 628-633, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays

Gerd Haberhausen,* Judith Pinsl, Carl-Christoph Kuhn, and Christine Markert-Hahn

Department of New Technologies, Laboratory Systems, Boehringer-Mannheim GmbH, D-82372 Penzberg, Germany

Received 7 July 1997/Returned for modification 3 October 1997/Accepted 18 November 1997

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5'-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.

* Corresponding author. Mailing address: Boehringer-Mannheim GmbH, Department LP-DN, Nonnenwald 2, D-82372 Penzberg, Germany. Phone: 49 8856 602666. Fax: 49 8856 602819. E-mail: gerd_haberhausen{at}bmg.boehringer-mannheim.com.

Journal of Clinical Microbiology, March 1998, p. 628-633, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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