Stable and noncompetitive RNA internal control for routine clinical diagnostic reverse transcription-PCR

Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G+C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.