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Journal of Clinical Microbiology, September 2004, p. 4130-4136, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4130-4136.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of the MagNA Pure LC Automated Nucleic Acid Extraction System followed by Real-Time Reverse Transcription-PCR for Ultrasensitive Quantitation of Hepatitis C Virus RNA

Linda Cook,1,2 Ka-Wing Ng,1,2 Arthur Bagabag,1,2 Lawrence Corey,1,2,3 and Keith R. Jerome1,2*

Departments of Laboratory Medicine,1 Medicine, University of Washington Medical Center,3 Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington2

Received 6 February 2004/ Returned for modification 28 March 2004/ Accepted 22 May 2004

Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay for HCV RNA. The assay amplifies a portion of the 5' untranslated region of HCV, which is then quantitated using the TaqMan 7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche). Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least 109 IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible, with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively. The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for routine HCV viral load testing.

* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D3-100, Seattle, WA 98109. Phone: (206) 667-6793. Fax: (206) 667-4411. E-mail: kjerome{at}fhcrc.org.

Journal of Clinical Microbiology, September 2004, p. 4130-4136, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4130-4136.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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