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Clinical Chemistry 45: 2079-2085, 1999;
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(Clinical Chemistry. 1999;45:2079-2085.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Ribonuclease-resistant RNA Controls (Armored RNA) for Reverse Transcription-PCR, Branched DNA, and Genotyping Assays for Hepatitis C Virus

Cindy R. WalkerPeach1,a, Matthew Winkler2, Dwight B. DuBois1 and Brittan L. Pasloske2

1 Cenetron Diagnostics LLC, 1834 State Highway 71 West, Cedar Creek, TX 78612.

2 Ambion, Inc., 2130 Woodward St., Suite 200, Austin, TX 78744.
a Address correspondence to this author at: Stratagene Cloning Systems, 1834 State Highway 71 West, Cedar Creek, TX 78612. Fax 512-321-3128; e-mail cindy_walkerpeach{at}stratagene.com

Background: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA® technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences.

Methods: A consensus 412-base sequence from the 5'NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat.

Results: AR-HCV-2b was fully recoverable from human plasma incubated at 4 °C for >300 days. The particles were tested in three clinical assay formats: AmplicorTM HCV Monitor 1.0, QuantiplexTM HCV RNA 2.0, and INNO-LiPATM HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay.

Conclusion: The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.




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