Improvement of HRV quantification using cRNA-based standards for real time RT-PCR

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.