Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript

Real-time PCR is a significant improvement over viral isolation and immunofluorescence for routinely detecting respiratory viruses. We developed three real-time internally controlled multiplex RT-PCR assays for detecting nine respiratory viruses.An internal control transcript consisting of a chimeric plasmid was synthesised and incorporated into each multiplex to monitor amplification efficiency, including inhibition. Each multiplex assay was developed on the Rotor-Gene 3000 and evaluated using RNA extracts from 126 nasopharyngeal aspirates from 112 pre-term infants.All 44/126 (35%) samples positive by immunofluorescence were confirmed by multiplex RT-PCR. Additionally, respiratory syncytial virus RNA was detected in 5 samples, influenza A virus RNA in 2 samples and thirteen (10%) dual infections by multiplex RT-PCR were noted. Inclusion of the RNA internal control did not affect the amplification efficiency of the target sequences and only 2 of 1256 (0.2%) samples tested over a 12 month period were inhibitory.Together with the improved sensitivity of the internally controlled multiplex RT-PCR assays over the older technology and the ability to detect co-infections, the internal control monitored the efficiency of both the RT and PCR steps and indicated inhibition, saving time and costs on running duplicate samples with a “spiked” inhibition control.

► We describe a multiplex RT-PCR that simultaneously detects nine respiratory viruses.
► An RNA internal control is used to monitor all aspects of detection and analyses.
► The internal control is produced as and when required using standard cloning methods.
► Unlike immunofluorescence, the multiplex RT-PCR can detect multiple infections.