Thermofisher taqpath 1 step rt qpcr master mix

Unless otherwise stated, RT-qPCR was conducted by applying a CFX96 Touch real-time PCR detection system (Biorad, Hercules, CA, USA) using 2.5 µL of the eluate, 2.5 µL Thermofisher TaqPath™ 1-Step RT-qPCR master mix (Thermo Fisher Scientific, Rochester, NY, USA) and 0.25 µL LightMix Modular Wuhan CoV RdRP-gene primer mix (cat-No. 53-0777-96, Tib Molbiol, Berlin, Germany) in a final reaction volume of 10 µL. An initial step of reverse transcription at 50 °C for 10 min followed by Taq activation at 95 °C for 5 min and 45 cycles of 95 °C 5 s, 60 °C 15 s and 72°C 15 s was performed.

RT-PCR testing was carried out within 24 h of sample collection by the Molecular Diagnostic Laboratory of LSU, an ISO 15189 and ISO 15190 accredited facility, and results were submitted to the national database. The test was performed targeting SARS-CoV-2 N1 and N2 genes, with human RNaseP as assay control target, after standard RNA extraction by MagMAX™ Viral/Pathogen II (MVP II) nucleic acid isolation kit using King Fisher Flex high-throughput automated extraction system (Thermo Fisher Scientific, Waltham, MA, USA). Primers recommended by Center for Disease Control (CDC) were used for the RT-PCR assay. In brief, we used Thermofisher TaqPath™ 1-Step RT-qPCR mastermix (Thermo Fisher Scientific, Waltham, MA, USA), no ROX. following a developed multiplex RT-PCR program on an ABI 7500 Fast DX instrument (Thermo Fisher Scientific, Waltham, MA, USA). 20 μL mastermix containing 5 μL of template was used for each test. The reaction condition includes steps of cDNA synthesis (10 min/55 °C), a hold step (1 min/95 °C), and subsequently 45 cycles of denaturation (10 s/95 °C) and annealing/elongation (30 s/55 °C) [23 (link)]. The threshold for a positive outcome was considered 40 amplification cycles.