Ribonuclease-resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus.
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 degrees C for >300 days. The particles were tested in three clinical assay formats: Amplicor HCV Monitor 1.0, Quantiplex HCV RNA 2.0, and INNO-LiPA 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.


CONCLUSIONS
The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.
WalkerPeach
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Cenetron Diagnostics LLC, 1834 State Highway 71 West, Cedar Creek, TX 78612, USA. cindy_walkerpeach@stratagene.com
Winkler
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DuBois
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Pasloske
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D001483:Base Sequence; D002138:Calibration; D004279:DNA, Viral / Q000032:analysis / Q000097:blood / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D004279:DNA, Viral; D007202:Indicators and Reagents; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic