A simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses

Laboratories often have difficulties obtaining positive control material for polymerase chain reaction (PCR) diagnosis of rare or emerging viruses. This is particularly problematic during outbreaks caused by emerging infectious diseases, when delays can impede the public health response.

The aim of this study was to develop a simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses.

We describe a universal method for preparing PCR positive reaction controls (Uni-Control), which uses two synthetic control oligonucleotides and irrelevant viral nucleic acid as an initiator template. In this study, we prepared Uni-Controls for novel influenza A(H1N1) and human metapneumovirus (HMPV) RT-PCR assays. Parainfluenza type 2 virus RNA and equine herpes virus DNA were used as initiator templates.

Using the Uni-Controls, characteristic sigmoidal real-time PCR amplification curves were observed in the influenza A(H1N1) and HMPV RT-PCR assays. Comparable cycle threshold values were observed in both assays when using the same concentration of the initiator template.

The Uni-Control method for preparing real-time PCR positive reaction controls provides an interim measure by which real-time PCR assays can be rapidly introduced for rare or emerging viruses in the absence of wild-type control material. The system is versatile and we propose can readily be adapted to almost any viral template.