RT-PCR for detecting the SARS-CoV-2 virus


1. Overview of the protocol

IMPORTANTE

The Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV is CE-IVD, FDA-approved on the Applied Biosystems™ 7500 Real-Time PCR System when following the manufacturer's product booklet. This online protocol below offers guidance on how to perform a control experiment and how to adapt the kit for different devices to support the scale-up effort of COVID-19 testing. These recommendations and alterations are therefore not covered by the CE-IVD, FDA validation of this kit.

Introduction

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.

Compatibility of this protocol

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.

Performance characteristics

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.

Limitations of the assay

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.

RNA extraction and QC

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.

2. Equipment and consumables

Material
  • RNA extracted from nasopharyngeal swabs or BAL, 10 μl per sample
  • Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV (SARS-CoV-2)

Consumibles
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml PCR strip tubes
  • RT-PCR-compatible 96-well plates (optical plates recommended)
  • RT-PCR-compatible 96-well plate lids or seals
  • Nuclease-free pipette filter tips
  • Highly recommended: Twist Biosciences Synthetic SARS-CoV-2 RNA Control 2 (cat # MN908947.3)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)

Instrumental
  • Applied Biosystems™ StepOnePlus™ Real-Time PCR System
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Microfuge
  • Mezclador vórtex
  • Cubeta con hielo
  • P1000 pipette
  • P200 pipette
  • P100 pipette
  • P20 pipette
  • P10 pipette
  • P2 pipette

Kit contents

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

Storage and shelf life

  • 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
IMPORTANTE

Reagents contained within a kit are intended to be used together. Do not mix reagents from different kit lots.

PCR plates

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:

PCR-plastic

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.

RNA extraction and QC

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.
IMPORTANTE

Standard curve

We recommend running a dilution series of a known RNA sample on your first plate to generate a standard curve. For subsequent experiments, you can use several diluted Positive Controls to account for inter-plate variability.

To make the standard curve, we recommend using Twist Biosciences’ Synthetic SARS-CoV-2 RNA Control. This is a synthetic SARS–CoV-2 RNA control that is supplied at a concentration of 1 million copies per µl. This is a higher concentration than the BGI Positive Control, and allows absolute quantification.

3. Reagent preparation

Material
  • Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV (SARS-CoV-2)

Consumibles
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml PCR strip tubes
  • Nuclease-free pipette filter tips

Instrumental
  • P1000 pipette
  • P200 pipette
  • P100 pipette
  • P20 pipette
  • Cubeta con hielo
  • Microfuge
IMPORTANTE

Standard curve

We recommend running a dilution series of a known RNA sample on your first plate to generate a standard curve. For subsequent experiments, you can use several diluted Positive Controls to account for inter-plate variability.

To make the standard curve, we recommend using Twist Biosciences’ Synthetic SARS-CoV-2 RNA Control. This is a synthetic SARS–CoV-2 RNA control that is supplied at a concentration of 1 million copies per µl. This is a higher concentration than the BGI Positive Control, and allows absolute quantification.

IMPORTANTE

The three stages of the protocol must be carried out in separate laboratory areas:

1st area: Reagent preparation 2nd area: Processing samples and controls 3rd area: RT-PCR
All materials used in one area should always remain in the area, and should not be moved to or used in other areas. Use separate boxes of pipette filter tips for each area.
After the assay is complete, the workbench and laboratory equipment should be cleaned and disinfected using 75% ethanol or UV light.
All used tubes and pipette tips must be discarded into a waste bin with bleach, and disposed of with other laboratory waste after decontamination.

Calculate the number of samples (N) in the test, which includes a blank control, a positive control, and the RNA samples (or serial dilutions of the RNA standard). We recommend each sample to be run in triplicate.

Prepare sufficient PCR tube strips for N reactions in the 1st laboratory area (reagent preparation area).

Thaw the Reaction Mix, Positive Control and Blank Control tubes at room temperature. Mix the tube contents briefly by vortexing and spin down the tubes. Store the reagents on ice.

Mix the contents of the Enzyme Mix by flicking the tube, and spin down briefly. Store immediately on ice.

In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare sufficient PCR master mix for N reactions. We recommend each sample and control to be run in triplicate.

Reagent Volume
Reaction Mix 18.5x 1.15N µl
Enzyme Mix 1.5x 1.15N µl
Total 20x 1.15N µl

This is sufficient for N reactions done in triplicate, with an additional 15% dead volume for each sample to account for pipetting error.

Mix gently by flicking the tube, and spin down.

Pipette 65 µl of PCR master mix into each PCR tube in the strips. Ensure no foam or bubbles form in the tubes.

Close the caps on the tubes, put them on ice, and transfer them to the 2nd laboratory area (sample and control processing area).

Immediately return the remaining Reaction Mix and Enzyme Mix to -20°C.

4. Processing samples and controls

Material
  • RNA extracted from nasopharyngeal swabs or BAL, 10 μl per sample
  • Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV (SARS-CoV-2)

Consumibles
  • Highly recommended: Twist Biosciences Synthetic SARS-CoV-2 RNA Control 2 (cat # MN908947.3)
  • RT-PCR-compatible 96-well plates (optical plates recommended)
  • RT-PCR-compatible 96-well plate lids or seals
  • 0.2 ml PCR strip tubes
  • Nuclease-free pipette filter tips
  • Nuclease-free water (e.g. ThermoFisher, AM9937)

Instrumental
  • P100 pipette
  • P10 pipette
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Microfuge
IMPORTANTE

This step must be carried out in the 2nd laboratory area for processing samples and controls.

Thaw the RNA extracted from swabs or BAL on ice. Mix the tubes by flicking, then spin down and return to ice.

MEDIDA OPCIONAL

Prepare your RNA standard dilution series.

Follow these instructions if you are making a dilution series of the Synthetic SARS-CoV-2 RNA Control 2:

  1. Thaw the RNA control on ice, mix by flicking the tube, briefly spin down and return to ice.
  2. Prepare an 8-tube PCR strip, and label seven tubes (e.g. S1-S7).
  3. Add 45 µl of nuclease-free water to each of the seven tubes, and close the lids.
  4. Take 5 µl of the RNA standard and transfer to tube S1. Close the lid, flick the tube to mix thoroughly and spin down.
  5. Take 5 µl of dilution S1 and add to tube S2. Close the lid, flick to mix and spin down.
  6. Repeat for all of the tubes up to and including tube S6 in the dilution series.
  7. Leave tube S7 with only nuclease-free water as a negative control.

Serial dilution

Add 32.5 µl each of the Blank Control, RNA samples (or the standard RNA dilution series), and Positive Control to the PCR strip tubes containing the PCR master mix. Pipette the samples directly into the master mix and avoid the samples touching the tube walls.

IMPORTANTE

To avoid contamination from exogenous RNA, add the samples in the following order: Blank Control, extracted RNA sample, Positive Control.

Close the tube lids, and label the exact location of controls and every sample.

Mix gently by flicking the tubes, and spin down.

Pipette 30 µl of each sample, in triplicate, into an RT-PCR-compatible 96-well plate.

Seal the plate, or close it with a lid.

Spin down the plate at 2,000 rpm for 10 seconds.

Label the location of all the samples and controls on the 96-well plate, and transfer it to the 3rd laboratory area (for RT-PCR).

Autoclave the used PCR tubes it in a sealed plastic bag.

5. RT-PCR (Applied Biosystems™ StepOnePlus™)

Instrumental
  • Applied Biosystems™ StepOnePlus™ Real-Time PCR System
IMPORTANTE

These instructions are written for the Applied Biosystems™ StepOnePlus™ Real-Time PCR System. If you are using a different system, please refer to the manufacturer's instructions for setting up your analysis.

Place the PCR plate into the thermal cycler.

Set up the Experiment Properties.

We recommend the settings displayed in the screenshot. If you are using thin-walled PCR plates, choose the Fast setting. Otherwise (or if you are unsure about which plates you have) the Standard setting is recommended.

RT-PCR 1

Set the fluorescence channels in the Plate Setup tab.

  • FAM channel (Reporter: FAM, Quencher: none) for SARS-CoV-2 RNA
  • VIC/HEX channel (Reporter: VIC/HEX, Quencher: none) for internal reference

Please refer to the manufacturer's instructions for detailed information on channel settings.

RT-PCR 2

Assign your samples.

Click the Assign Targets and Samples button at the bottom of the Plate Setup page. Follow the instructions at the top of the tab to define the position of your samples and controls.

RT-PCR 5

MEDIDA OPCIONAL

Set up your standard curve.

If you are using the Twist Biosciences Synthetic SARS-CoV-2 RNA Control 2 to set up a standard curve, click the Define and Set up Standards button on the Plate Setup page. Set the well positions and details of the standard curve.

RT-PCR 4

Set the program in the Run Methods tab.

Set the program shown below. Also set the reaction volume to 30 µl.

RT-PCR 4.2

Step Cycle Temperature Duration Fluorescence measured? (Y/N)
1 1 cycle 50°C 20 min N
2 1 cycle 95°C 10 min N
3 40 cycles 95°C
60°C
15 sec
30 sec
N
Y

Start the program on the thermal cycler.

After the RT-PCR reaction is complete, remove the 96-well plate from the thermal cycler and autoclave it in a sealed plastic bag.

Clean all the workbenches and laboratory equipment/supplies, and decontaminate the area with 75% ethanol or UV light.

6. RT-PCR data analysis

Quality control

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. Negative control
  • Positive Control: The standard curves for the FAM and VIC/HEX channels are S-shaped, with Ct values not higher than 32. Positive control FAM Positive control VIC
  • RNA specimen: The standard curves for the VIC/HEX channels are S-shaped, with Ct values not higher than 32. RNA specimen

Threshold and reference range

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.

Standard curve

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

Twist Std Curve

Twist Std Curve2

Interpreting results

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.

Last updated: 4/30/2020

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