Strategic approach to produce low-cost, efficient and stable competitive internal controls for the detection of RNA viruses using RT-PCR

VILLANOVA, GABRIELA VANINA,; GARDIOL, DANIELA; TABORDA, MIGUEL A.; REGGIARDO, VIRGINIA; TANNO, HUGO; RIVADENEIRA, EMILIA D.; PEREZ, GERMÁN R.; GIRI, ADRIANA

JOURNAL OF CLINICAL MICROBIOLOGY

American Society for Microbiology

Lugar: Washington, DC; Año: 2007 vol. 45 p. 3555 - 3563

0095-1137

Molecular diagnostics based on RT-PCR routinely is 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 (rQß) bearing KY78/80 primers that are widely used in the detection of hepatitis C virus (HCV). The Qß genome was previously cloned as cDNA in an expression vector under the T7 promoter control (pBRT7Qß). A recombinant Qß genome was generated by PCR using a pBRT7Qß template into with 50 bp primers containing KY78 or KY80, phage sequences and restriction sites had been included. The final construct (pBRT7rQß) was confirmed by sequencing. BL21 cells were transformed with pBRT7rQß and, after induction with IPTG, rQß lysates were obtained. rQß was RNase-resistant and stable at 4°C for 452 days in SM medium 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 non-radioactive probes and enzyme immunoanalysis. No influence of the CIC on 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 the armored RNA technology that can be adapted for detection or real time applications of any RNA target. Moreover, the CIC reported here is an essencial reagent for HCV screening in blood banks in resource-limited settings. KEY WORDS: competitive internal control, RT-PCR, recombinant Qß coliphage, RNA viruses, HCV