Strategic approach to produce low-cost, efficient and stable competitive internal controls for detection of RNA viruses by use of reverse transcription-PCR.

VILLANOVA, G. V.; GARDIOL, DANIELA; TABORDA, MIGUEL A.; REGGIARDO, V.; TANNO, H; RIVADENEIRA, E.D.; PEREZ, G. R.; GIRI ADRIANA ANGELICA

JOURNAL OF CLINICAL MICROBIOLOGY

American Society for Microbiology

Año: 2007 vol. 45 p. 3555 - 3563

0095-1137

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 QB phage derivative (recombinant QB [rQB] earing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQB was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO4
7H2O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQB performance as a CIC was evaluated. rQB 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.