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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

AN Aidan M. Nikiforuk
TC Todd A. Cutts
ST Steven S. Theriault
BC Bradley W. M. Cook
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RNA was extracted from viral supernatant using commercial reagents (Viral RNA Isolation Kit, Qiagen) and quantified by qRT-PCR (Lightcycler 480 RNA Master Hydrolysis Probes, Roche). Ebola virus RNA was detected by use of a 2:1 primer probe mix (300 nm, ZEBOV LF- CAGCCAGCAATTTCTTCCAT, ZEBOV LR-TTTCGGTTGCTGTTTCTGTG: 150 nm, ZEBOV LP1 -6FAM’-ATCATTGGCGTACTGGAGGAGCAG’MGBNFQ, ZEBOV LP2- 6FAM’-TCATTGGCGTACTGGAGGAGCAGG’MGBNFQ) specific for Zaire ebolavirus species. The conditions of the PCR reaction were programmed to: 61 °C for 3 min, 95 °C for 30 sec, 40 cycles of 95 °C for 15 sec and 60 °C for 40 sec. VSV-GFP RNA was detected by using a primer probe set targeting the nucleoprotein region (VSV-GFP NPF- CGGAGGGACGACTAATGC, VSV-GFP NPR-CGAGCGATTCGACCACATC, probe-6FAM’CGCCACAAGGCAG’MGBNFQ). The same thermo-cycling conditions were used.

Copyright and License information: The Author(s) ©2017
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