This protocol is for an in vitro nucleic acid amplification assay to detect the novel SARS-CoV-2 virus using reverse transcription real-time PCR from nasopharyngeal swabs or bronchoalveolar lavage (BAL) fluid.

The kit is based on in vitro RT-PCR combining fluorescent probing. Primers and sequence-specific fluorescence probes were designed to target a highly-conserved region in the SARS-CoV-2 genome. The probes are oligonucleotide-attached fluorophores with a 5' FAM reporter and a 3' quencher. Additionally, specific primers and probes were developed as an internal reference with VIC/HEX fluorophores attached at the 5' end as a reporter. During the PCR procedure, when the probes hybridise to the target DNA, the DNA polymerase cleaves the probe at the 5' end and separates the reporter dye from the quencher. This cleavage results in a fluorescent signal generated by the cleaved reporter dye, which is monitored in real-time by the PCR detection system. Monitoring the fluorescence intensities during RT-PCR allows the qualitative detection of the SARS-CoV-2 sequence in samples.

This kit has been used with the Applied Biosystems™ StepOnePlus™ Real-Time PCR System. When other systems are considered, the user will need to follow the manufacturer's instructions for set-up. If you have validated this protocol on a different RT-PCR system and would like to share your results, please contact Support.

The kit packaging is intact and the tube contents are clear, transparent, and with no sediment. All the contents are in the correct quantity, as described in the kit insert and in this protocol.

The Positive Control is positive in both the FAM and VIC/HEX channels, while the Blank Control is negative in both channels.

The limit of detection (LOD) of the kit is 100 copies/ml for SARS-CoV-2.

Potential cross-reactivity of the kit was tested, and none of the tested pathogens have been reactive. The test included human coronaviruses: HCoV-OC43, HCoV-229E, HCoV-HKU1 and HCoV-NL63.

This kit is for in vitro use only.

Please read this protocol carefully before starting the test. Sample collection, storage and transportation, and the laboratory test should be carried out strictly in line with relevant biosafety and laboratory management regulations.

The results of the test are intended as information for clinical practices to assess the infection status of patients, and should be combined with clinical presentations and other laboratory markers.

A false positive or negative result can be caused by incorrect sample collection, transportation and processing, very low concentration of the target virus in the sample, mutations in regions of the viral genome that are covered by the kit primers or probes, and external factors such as PCR inhibitors. The operator should have a good understanding of the principles of the procedure as well as its limitations to avoid any potential mistakes.

For extracting RNA from nasopharyngeal swabs or BAL, we recommend using the MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit. The RT-PCR reaction requires 10 μl of extracted RNA per sample. The RNA has to pass the minimum QC criteria:

• The OD 260/280 value (as measured by the Nanodrop spectrophotometer) should be ~2, and the 260/230 value should be 1.8–2.2.
• The RNA Integrity Number (RIN, as measured using the Agilent Bioanalyser) should be 7 or above.

Each kit provides sufficient reagents for 50 reactions.

Contents	Volume	Quantity	Description
Reaction Mix	1 ml/vial	1 vial	Amplification reagents, probes and primers
Enzyme Mix	80 µl/vial	1 vial	Taq polymerase, reverse transcriptase and UDG
Positive Control	750 µl/vial	1 vial	Mixed solution of recombinant plasmids containing target virus genes and internal reference
Blank Control	750 µl/vial	1 vial	Nuclease-free water

• The RT-PCR kit should be stored at -20°C in a dark place
• The kit is stable for 5 days at 2-8°C and for 6 months at -20°C
• If the kit is transported, it should be kept at -20°C in the dark
• Avoid freeze-thawing the unopened kit more than 4 times

Plastic plates or tubes used in qPCR can influence the PCR reaction. In this period of short consumables supply, users should ensure they understand the thickness of the plastic and the colour to appropriately configure their qPCR instruments.

Colour qPCR plastics are typically available with the following surfaces: Optical, frosted, or white. For machines where reading happens on the side or bottom of the well, optical-grade plastic is a requirement. Where the reader is on the top of the well, the following behaviours have been observed:

If you are changing to a different type of PCR plates (e.g. from optical to frosted or white) due to supply restrictions, ensure that you account for signal differences. We recommend running a control sample between the two plate types to ensure you re-calibrate your results.

Thickness qPCR plastics can be supplied as thin-walled or standard. The thickness of the plastic will impact your device settings: the thermal cycler will perform faster temperature ramps when using thin-walled tubes than thick-walled, and this will impact PCR performance.

If you are changing to a different type of PCR plates due to supply restrictions, ensure that you account for ramp speeds and verify that the correct settings are chosen. At Oxford Nanopore Technologies, we have found that running Fast cycling mode with thin-walled plates gave the best results. We recommend running a control sample between the two plate types to ensure you re-calibrate your results.

This protocol is a working version of what has been tried and tested, with notes about the optimisations made to different devices to accommodate for consumable changes.

Part number/vendor	Trial location	Trial qPCR device	Comments
MicroAmp™ Optical 96-Well Reaction Plate, ThermoFisher, N8010560	Oxford Nanopore Technologies	Applied Biosystems™ StepOnePlus™	When running the machine in Standard PCR cycling settings, unusual amplification curves were observed. By setting the temperature ramp settings to Fast PCR, the results normalised and behaved as expected.

For extracting RNA from nasopharyngeal swabs or BAL, we recommend using the MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit. The RT-PCR reaction requires 10 μl of extracted RNA per sample. The RNA has to pass the minimum QC criteria:

• The OD 260/280 value (as measured by the Nanodrop spectrophotometer) should be ~2, and the 260/230 value should be 1.8–2.2.
• The RNA Integrity Number (RIN, as measured using the Agilent Bioanalyser) should be 7 or above.

The requirements listed below must be met for each test to be valid. Otherwise, repeat the test again adhering to the instructions in the protocol.

• Blank Control: Ct values for FAM and VIC/HEX channels are 0, or no data available.
• Positive Control: The standard curves for the FAM and VIC/HEX channels are S-shaped, with Ct values not higher than 32.
• RNA specimen: The standard curves for the VIC/HEX channels are S-shaped, with Ct values not higher than 32.

The cut-off value for the kit was determined based on the Receiver Operator characteristic curve from testing clinical samples. Ct values for SARS-CoV-2 positive samples should not be higher than 38.

The standard curve using the Twist Biosciences Synthetic SARS-CoV-2 RNA Control 2 should look similar to the graphs below.

For each sample and control, take the average Ct value for the three repeats. If one repeat differs significantly from the other two, discard this data point.

• The sample is positive for SARS-CoV-2 if the standard curve in the FAM channel is S-shaped and the Ct value of the sample is not higher than 38.
• The sample is negative for SARS-CoV-2 if the standard curve in the FAM channel is not S-shaped and the Ct value of the sample is 0 or no data is available, while the Ct value at the VIC/HEX channel is not higher than 32.
• The sample should be retested if the standard curve in the FAM channel is S-shaped and the Ct value of the sample is higher than 38. Following retesting, the sample can be reported as positive for SARS-CoV-2 if the Ct value is higher than 38, and as negative if the standard curve is not S-shaped and the Ct of the VIC/HEX channel for the internal reference is not higher than 32.
• The sample should also be retested if the standard curve in the FAM channel is not S-shaped and the Ct value of the sample is 0 or no data available, and the Ct value in the VIC/HEX channel is higher than 32 or no data is available.