Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

Abstract

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.

Analytical Methods for Food and Environmental Viruses

“…The mengovirus (Comelli et al 2008;Costafreda et al 2006;da Silva et al 2007) and the feline calicivirus (Mattison et al 2009a) have been proposed as sample process controls for food and environmental virology samples. Many control systems assess only the final detection step and evaluate potential inhibition (Casas et al 2007;Dingle et al 2004;Escobar-Herrera et al 2006;Hoorfar et al 2003;Hoorfar et al 2004;La Rosa et al 2007;Medici et al 2005). It is possible to detect an internal amplification control even though the extraction procedure has failed, for example by an error that reduces the quality of the extracted RNA.…”

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

“…Noncompetitive IPC, however, uses a completely separate target that does not directly compete with the target amplicon and has a different set of primers and probe not shared with the target amplicon for its amplification. The IPC for RRT-PCR can be introduced in a variety of ways, including transcribed RNA, armored RNA, inactivated virus, or plasmid DNA (1,6,9,10,12,14,19,27,28,31). In all cases, the IPC can be monitored in the same reaction by resolving the products on an agarose gel or using different fluorescent probes in a multiplex format with RRT-PCR.…”

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

“…In the noncompetitive IC strategy, separate primer pairs are used to detect the control and the target of interest. This approach is attractive because a noncompetitive IC could be used as a universal control for the detection of different RNA targets (6,7). However, amplification of such a control may not accurately reflect the amplification of the target due to differences in the amplification efficiencies between them.…”

“…For these purposes, noncompetitive ICs and competitive ICs (CICs) have been described (2,5,6,7,8,9,14,25,32). In the noncompetitive IC strategy, separate primer pairs are used to detect the control and the target of interest.…”