TITLE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR N gene 2020 
AUTHORS: Judy Northill, Ian Mackay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">A real-time RT-PCR to designed to detect SARS-CoV-2 and other related sarbecoviruses. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. </li><li style = "counter-reset:ol0;list-style-type:disc;">The target region encodes the nucleocapsid (N).</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Tested on wild-type SARS-CoV-2 virus , it is expected to be capable of detecting SARS-CoV-2, bat-like SARS and SARS virus (members of the subgenus </span><span style = "font-style:italic;">Sarbecovirus</span><span>).</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Limit of detection not yet determined.</li><li style = "counter-reset:ol0;list-style-type:disc;">The performance of the assay has not been tested with low viral load samples or samples from patients who are clinically well.</li><li style = "counter-reset:ol0;list-style-type:disc;">The sensitivity of  this assay was improved with the use of the SensiFast™Probe Lo-ROX One-step kit. </li><li style = "counter-reset:ol0;list-style-type:disc;"><span>A single 1 mismatch at probe-binding site identified with the </span><span style = "font-style:italic;">BetaCoV/USA/CA1/2020|EPI_ISL_406034</span><span>  (GenBank MN994467.1) variant of SARS-CoV-2 (as of 23JUNE2020).</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Probe is in the 3'-5' (reverse complement) direction.</li><li style = "counter-reset:ol0;list-style-type:disc;">Reverse primers were replaced in March 2020.</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>We also recommend the ORF1ab assay (</span><a href="https://dx.doi.org/10.17504/protocols.io.bgtmjwk6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020</span></a><span>), US-CDC-N1 assay or the E gene assay by Corman </span><span style = "font-style:italic;">et al</span><span>. (</span><a href="https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Protocol v2-1</span></a><span style = ":;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Notes:  </span></li><li style = "counter-reset:ol0;list-style-type:disc;">Assay is optimised.</li><li style = "counter-reset:ol0;list-style-type:disc;">This test has identified clinical positive cases of coronavirus disease (COVID-19)</li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Notes:  </span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Assay is optimised.</li><li style = "counter-reset:ol0;list-style-type:disc;">This test has identified clinical positive cases of coronavirus disease (COVID-19)</li></ul></div></div>

[STEPS]
?. [Mix]
Oligonucleotides  ABC1Oligo NameSequence 5'-3'Location based on NC_045512.2*2Wuhan-TM2020ForTCGTGCTACAACTTCCTCAAG28743-287633Wuhan-TM2020Probe6FAM-CCGCCTCTGCTCCCTTCTGC-BHQ128809-287904SARS2-28875R-GCTGCCTGGAGTTGAATTTCTTG28875-288545SARS2-28875R-A CTGCCTGGAGTTGAATTTCTTA 28875-28854
ABC1Oligo NameSequence 5'-3'Location based on NC_045512.2*2Wuhan-TM2020ForTCGTGCTACAACTTCCTCAAG28743-287633Wuhan-TM2020Probe6FAM-CCGCCTCTGCTCCCTTCTGC-BHQ128809-287904SARS2-28875R-GCTGCCTGGAGTTGAATTTCTTG28875-288545SARS2-28875R-A CTGCCTGGAGTTGAATTTCTTA 28875-28854
?. [Mix]
Reagents
?. [Mix]
Synthetic controlsSynthetic controls are produced using the binary synthetic template oligonucleotide positive control for in-house diagnostic real-time RT-PCR method.The oligonucleotide sequences required to make controls for this assay are:Probe control: AAAATAATACGACTCACTATAGGGTGAAGAGAATCCACAAGGAATTGAACCGCCTCTGCTCCCTTCTGCACAGTGTTCAGCAGGTCCTGTTGAAAAPrimer control:AAAATAATACGACTCACTATAGGGTCGTGCTACAACTTCCTCAAGATGATCTGGCACGGGACCCTCCAAYAAGAAATTCAACTCCAGGCAGAAAA
?. [Mix]
Reaction Set-upAssay has been designed to be used on both a Rotor-Gene 6000 / Rotor-Gene Q 5-plex using 100-place rotor discs and an ABI 7500 Fast real-time machine.Total reaction volume is 20µL.Prepare sufficient for number of reaction plus a 'dead volume' usually 2 extra. Adjust as necessary if using a robotic dispenser.  ABC1ReagentVolume (µL) x1Final reaction concentration2Nuclease free water4.23Wuhan-TM2020F (200µM)0.05500nM4SARS2-28875R-G (200µM)0.05500nM5SARS2-28875R-A (200µM)0.09900nM6Wuhan-TM2020Probe (100µM)0.0150nM72 X SensiFast Probe Lo-Rox One-Step mix*101X8RiboSafe RNase Inhibitor*0.49Reverse transcriptase*0.210TOTAL VOLUME15Dispense 15µl to each reaction well.Add 5µl of template, extracted RNA, controls or NTC (nuclease-free water).Total reaction volume is 20µl.
ABC1ReagentVolume (µL) x1Final reaction concentration2Nuclease free water4.23Wuhan-TM2020F (200µM)0.05500nM4SARS2-28875R-G (200µM)0.05500nM5SARS2-28875R-A (200µM)0.09900nM6Wuhan-TM2020Probe (100µM)0.0150nM72 X SensiFast Probe Lo-Rox One-Step mix*101X8RiboSafe RNase Inhibitor*0.49Reverse transcriptase*0.210TOTAL VOLUME15
?. [Amplification]
PCR amplification  AB11 cycle40 cycles250°C     5min95°C 3 seconds395°C     2min60°C 30 seconds*
AB11 cycle40 cycles250°C     5min95°C 3 seconds395°C     2min60°C 30 seconds*
?. [Result Analysis]
The definition used for a satisfactory positive result from a real-time fluorogenic PCR should include each of the following:A sigmoidal curve – the trace travels horizontally, curves upward, continues in an exponential rise and followed by a curve towards a horizontal plateau phaseA suitable level of fluorescence intensity as measured in comparison to a positive control (y-axis)A defined threshold (CT) value which the fluorescent curve has clearly exceeded (Fig.1 arrow) and which sits early in the log-linear phase and is A flat or non-sigmoidal curve or a curve that crosses the threshold with a CT value >40 cycles is considered a negative resultNTCs should not produce a curve Figure 1. Examples of satisfactory sigmoidal amplification curve shape when considering an assay’s fluorescent signal output. The crossing point or threshold cycle (CT) is indicated (yellow arrow); it is the value at which fluorescence levels surpass a predefined (usually set during validation, or arbitrary) threshold level as shown in this normalized linear scale depiction. LP-log-linear phase of signal generated during the exponential part of the PCR amplification; TP-a slowing of the amplification and accompanying fluorescence signal marks the transition phase; PP-the plateau phase is reached when there is little or no increase in fluorescent signal despite continued cycling.