TITLE: Multiplex SARS-CoV-2 RT-qPCR protocol
AUTHORS: Peter H L Krijger, Tim A Hoek, Sanne Boersma, Lieke I P M Donders, Maaike M C Broeders, Mark Pieterse, Pim W Toonen, Ive Logister, Bram M P Verhagen, Marjon J A M Verstegen, Thomas W van Ravesteyn, Rene J T M Roymans, Francesca Mattiroli, Jo Vandesompele, Monique Nijhuis, Stefan Meijer, Anton van Weert, Edwin Dekker, Fred J Dom, Rob Ruijtenbeek, Lieven B J van der Velden, Jeroen H B van de Bovenkamp, Martijn Bosch, Wouter de Laat, Marvin E Tanenbaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>An in-house multiplex RT-qPCR , targeting SARS-CoV-2and PDV as internal control [1][2], developed on QuantStudio 7 Pro Real-Time PCR Systems using Life Technologies Taqman FastVirus 1-step mastermix with E-gene primers and probe as described by Corman </span><span style = "font-style:italic;">et al.</span><span> and N1 primers and probes as described by the CDC[3, 4].</span></div><br/><div class = "text-block"><span>1.Clancy, A. eta al., </span><span style = "font-style:italic;">The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus </span><span>European Journal Microbial Infectious Diseases, 2008. </span><span style = "font-weight:bold;">276</span><span>(12): p.1177.</span></div><div class = "text-block"><span>2.Wolters, F., et al., </span><span style = "font-style:italic;">Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic</span><span>. Journal of Clinical Virology, 2020. 128: p. 104426</span></div><div class = "text-block"><span>3.Corman, V.M., et al., </span><span style = "font-style:italic;">Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.</span><span> Euro Surveill, 2020. </span><span style = "font-weight:bold;">25</span><span>(3).</span></div><div class = "text-block"><span>4.Lu, X., et al., </span><span style = "font-style:italic;">US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2.</span><span>Emerging Infectious Disease journal, 2020. </span><span style = "font-weight:bold;">26</span><span>(8): p. 1654.</span></div></div>

[STEPS]
?. Run the RT-qPCR program:Time (s)Temp (°C)1x300551x209540x3953058
Time (s)Temp (°C)1x300551x209540x3953058
?. Prepare the RT-qPCR master mix: ReagentsVolume (1 reaction)4x TaqMan Fast Virus 1-Step Master Mix3.75 µlprimer/probe mix0.75 µlNuclease-free water  3.0 µlRNA7.5 µlTotal15 µl
ReagentsVolume (1 reaction)4x TaqMan Fast Virus 1-Step Master Mix3.75 µlprimer/probe mix0.75 µlNuclease-free water  3.0 µlRNA7.5 µlTotal15 µl
?. Reconstitute the oligos and probes to 100uM with nuclease-free water, vortex briefly and prepare the primer/probe mix:  100uM stock    Volume (1 reaction)     Volume (1000 reactions)    tm PDV F1 (100 nM/reaction)    0.015 µl  15 µl  tm PDV R1 (100 nM/reaction)    0.015 µl  15 µl  tm PDV P (100 nM/reaction)    0.015 µl  15 µl  2019-nCoV_N1-F1 (600 nM/reaction)    0.09 µl  90 µl  2019-nCoV_N1-R1 (600 nM/reaction)    0.09 µl  90 µl  2019-nCoV_N1-P (200 nM/reaction)    0.03 µl  30 µl  E_Sarbeco_F1 (400 nM/reaction)    0.06 µl  60 µl  E_Sarbeco_R2 (400 nM/reaction)    0.06 µl  60 µl  E_Sarbeco_P1 (200 nM/reaction)    0.03 µl  30 µl  Nuclease-free water    0.345 µl  345 µl  Total    0.75 µl  750 µlNote: The primer/probe mix can be prepared in advance, aliquoted in microcentrifuge tubes, and stored at -20°C for 1 year.
100uM stock    Volume (1 reaction)     Volume (1000 reactions)    tm PDV F1 (100 nM/reaction)    0.015 µl  15 µl  tm PDV R1 (100 nM/reaction)    0.015 µl  15 µl  tm PDV P (100 nM/reaction)    0.015 µl  15 µl  2019-nCoV_N1-F1 (600 nM/reaction)    0.09 µl  90 µl  2019-nCoV_N1-R1 (600 nM/reaction)    0.09 µl  90 µl  2019-nCoV_N1-P (200 nM/reaction)    0.03 µl  30 µl  E_Sarbeco_F1 (400 nM/reaction)    0.06 µl  60 µl  E_Sarbeco_R2 (400 nM/reaction)    0.06 µl  60 µl  E_Sarbeco_P1 (200 nM/reaction)    0.03 µl  30 µl  Nuclease-free water    0.345 µl  345 µl  Total    0.75 µl  750 µl