Straightforward Procedure for Internal Control of Real-Time Reverse Transcription Amplification Assays

Internal controls are crucial for reliable detection of pathogens in nucleic acid amplification assays. Without an internal control, a negative amplification result may mean that the sample contains no target or that the reaction failed.We recently described (1) the use of internal controls that consist of internal control oligonucleotides (ICOs) that contain little more than primer sites and a probe-binding site that contains mismatches to the actual target. ICOs can be obtained commercially and do not require a separate control-specific detection probe. Because of the mismatches, the control does not hybridize to the single detection probe during the fluorescence-acquisition step, and thus the probe binds and detects only nucleic acid derived from the pathogen. However, amplification of the ICO can be verified during melting analysis after PCR.Many important viral pathogens contain an RNA genome that must be transcribed to cDNA before amplification. To provide a control for both reverse transcription and amplification, an internal control must be an RNA molecule. The standard method for generating such a control involves directional cloning of a double-stranded DNA template into a plasmid vector carrying bacteriophage RNA polymerase promoter sequences. The RNA is then transcribed in vitro from the DNA template (2)(3).We have extended the ICO technique to RNA by introducing an RNA polymerase promoter and adding 2 simple preparation steps. We demonstrate the application of this control technique to the reverse transcription-PCR (RT-PCR) step of a LightCycler (Roche Molecular Biochemicals) real-time hybridization probe assay for respiratory syncytial virus (RSV).The sequences of the primers and probes for the RSV assay were 5′-GCCTTGTTTCCACAATA-3′, 5′-TCTTCATCACCATACTTTTCTGTTA-3′, 5′-CY5.5-GGAATTCACATGGTCTACTACTGACTGT-phosphate, and 5′-GTTGTTCTATAAGCTGGTATTGATGCA-fluorescein-3′ (4). The 2-step LightCycler (Roche) RT-PCR was performed as follows. For the reverse transcription step (total volume of 10 μL), the reaction mixture consisted of 1.5 mM MgCl2, 2.5 …