Figure 1. Reaction temperature profile for RapiDxFireThermostable Reverse Transcriptase. Reverse transcriptionreactions were set up on ice using each manufacturer’s recommended buffersystem and Poly (rC) /p(dG)12-18 template/primer substrate. Reactions weretransferred from ice to the indicated temperatures (37, 50, 55, 60, 65, 70, 75,and 80°C) and incubated for 40 minutes. Following incubation, RNA/cDNAheteroduplex product is quantified as a measure of polymerization activity,utilizing PicoGreen® fluorescence on a Tecan Infinite® M1000 Pro. RapiDxFire ThermostableRT exhibits increasing activity as the reaction temperature is increased, up to80ºC (highest temperature tested); ~60% activity remains after 10 min at 90⁰C(data not shown).
SuperiorDetection of Zika virus with RapiDxFire Thermostable RT(检测寨卡病毒的优异性)
Figure 2. cDNA synthesistime course studies in a 2-step RT-qPCR reaction with different thermostableRTs. A) qPCR curve after a 1 minute reversetranscription reaction. Zika cDNA synthesis was conducted for each enzyme induplicate using target-specific primers and recommended reaction buffer andincubation temperatures per suppliers’ guidelines (RapiDxFire= 60⁰C, SupplierB= 50⁰C, and Supplier T= 55⁰C). After cDNA synthesis real time PCR wasperformed using one-tenth volume of each of the cDNA samples. PCR was performedusing EconoTaq and its supplied buffer (Lucigen Cat No. 30031-3). Detection ofPCR products were done real time using an intercalating dye (Dyomics Cat No. V13-01184)and BioRad CFX C1000 Touch™ with absorption/emission of 481nm/526nm. B) Reversetranscription time course study prior to performing second-step real-time PCR.Encircled data points are derived from data represented in the qPCR curve inA).