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Journal of Clinical Microbiology, November 2007, p. 3555-3563, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.02601-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR

Área Virología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina,¹ Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Rosario, Argentina,² Cátedra de Gastroenterología, Hospital Provincial del Centenario, Rosario, Argentina,³ Division of Pediatric Infectious Diseases, Department of Pediatrics, Mount Sinai Medical Center, New York, New York⁴

Received 28 December 2006/ Returned for modification 11 April 2007/ Accepted 31 May 2007

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 Qß phage derivative (recombinant Qß [rQß]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQß 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. rQß performance as a CIC was evaluated. rQß 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.

Published ahead of print on 15 August 2007.