Qpcrbio 1 step go lo rox kit

A standard, non-nested, RT-qPCR protocol was used to determine the LLOQ plasma viral load of the assay. The qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems) was used with the same oligonucleotide and tRNA concentrations as in the qPCR protocol. As recommended by the manufacturer, an initial reverse transcription step of 50 °C for 10 min was followed by a “touch up” cycling protocol with an enzyme activation step of 95 °C for 2 min; 40 amplification cycles of 95 °C for 10 s, 52 °C → 60 °C* for 10 s and finally 72 °C for 1 min. The arrow and asterisk indicates that the annealing temperature was increased from 52 to 60 °C in equal increments across 40 cycles. Five µl of sample was used in a final reaction volume of 50 µl for maximum sensitivity.

For initial primer evaluation, the QuantiTect Probe kit (Qiagen) for qPCR of DNA targets and the SuperScript III Platinum One-Step qRT-PCR Kit (ThermoFisher Scientific) for RNA targets, were used because they were the reagents commonly used in our laboratories.
To shorten assay time using fast-cycling, we evaluated the use of the following test kits and master mixes for qPCR:—the QuantiFast Probe (Qiagen); Kapa Probe Fast (Merck) ;or qPCRBIO Probe Mix Lo-Rox (PCR Biosystems); the EXPRESS One-Step SuperScript (ThermoFisher Scientific); and the qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems), based on recommendations from PCR instrument manufacturers and colleagues.
To further increase assay sensitivity and reduce variability at low target concentrations, various concentrations of tRNA were added to the master-mix and optimized. A published “step-up” cycling protocol was employed to improve sensitivity and cross-subtype specificity³⁸ . “Step-up” cycling rescues PCR reactions where there are minor mismatches between the assay oligonucleotides and the target template species and serves to increase the sensitivity and precision for detection of HIV subtypes with minor sequence polymorphisms at low target concentrations.