Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR
| Autor: | Gabriela Vanina Villanova, Daniela Gardiol, Miguel A. Taborda, Emilia D. Rivadeneira, Hugo Tanno, Adriana A. Giri, Germán R. Perez, Virginia Reggiardo |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Microbiology (medical)
Base Sequence biology Reverse Transcriptase Polymerase Chain Reaction RNase P Hepatitis C virus Molecular Sequence Data RNA RNA virus Hepacivirus biology.organism_classification medicine.disease_cause Coliphages Sensitivity and Specificity Virology Molecular biology Reverse transcriptase Reverse transcription polymerase chain reaction medicine Humans RNA Viral RNA extraction Viral load |
| Zdroj: | Journal of Clinical Microbiology. 45:3555-3563 |
| ISSN: | 1098-660X 0095-1137 |
| DOI: | 10.1128/jcm.02601-06 |
| Popis: | Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO 4 ·7H 2 O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|