This protocol details how to make and use a pair of synthetic template oligonucleotides (STOs) for use as real-time reverse transcription polymerase chain reaction (RT-rPCR) controls. The method can also be used for PCR controls. This binary positive control approach is easy to replicate once mastered, quick to produce, results in enough RNA stock for years of PCR runs, and provides several benefits over the use of a wild-type positive control such as the nucleic acids extracted from virus culture or from a known previously-positive clinical specimen.The method is an adaptation of a previsouly published approach [Ref 1] and has recently been described in some publications.[Ref 2]Our approach to a positive control requires that each PCR run includes two additional reactions. one controls for primer function (PRIMER control). This STO includes hybridisation sites for the specific target assay's primers bouding a generic probe hybridisation target siteone controls for probe function (PROBE control). This STO includes a hybridisation site for the specific target assay's probe(s), bounded by a pair of generic primer hybridisation target sites REFERENCES.Smith G, Smith I, Harrower B, Warrilow D, Bletchly C. A simple method for preparing synthetic controls for conventional and real-time PCR for the identification of endemic and exotic disease agents. J Virol Methods. 2006 Aug;135(2):229-34. https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/16677717Hall-Mendelin S, Pyke AT, Moore PR, Mackay IM, McMahon JL, Ritchie SA, Taylor CT, Moore FA, van den Hurk AF. Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia. PLoS Negl Trop Dis. 2016 Sep 19;10(9):e0004959. https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/27643685Transcription of RNA templates by T7 RNA polymerase. Nucl. Acids Res. (1998) 26 (15): 3550-3554. Arnaud-Barbe et al.REFERENCES.