One-step NZYSpeedy RT-qPCR Probe kit, ROX plus is an optimized and highly efficient reaction mixture developed for first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit includes a One-step NZYSpeedy qPCR Probe master mix developed for fast PCR, provided as a 2× reaction mixture, which contains all components necessary for real-time PCR, including dNTPs, stabilizers, and enhancers. In addition, a separate NZYRT mix that comprises a balanced mixture of both Reverse transcriptase and Ribonuclease Inhibitor is also provided. This kit enables fast and highly reproducible procedures on the most common real-time PCR apparatus, from either total RNA or mRNA. It was developed for probe-detection technology, including molecular beacons. The latest developments in PCR enhancers have been incorporated in the One-step NZYSpeedy RT-qPCR Probe kit, ROX plus, including buffer chemistry and a polymerase with hotstart-like activity. This optimized mixture together with the NZYRT mix results in highly sensitive and successful results.