TITLE: SARS-CoV-2 detection using BGI RT-PCR kit
AUTHORS: Wei-Ting Lu, Ming Jiang, Robert Goldstone, Karen Ambrose, Chris Ekin, Amy Strange, Nnenna Kanu, Paul Grant, Efthymios Fidanis, Jerome Nicod, David Moore, Michael Howell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose of examination / Clinical relevance</span></div><div class = "text-block">At the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing. The first step of the process is sample receipt at the Crick.</div><div class = "text-block">This protocol describes the preparation of the RT-PCR master mix plate, automated addition of RNA template and the reverse-transcriptase quantitative PCR of viral RNA to detect the Orf1ab region of the viral genome, which has high specificity to the 2019-nCoV virus. Furthermore, the protocol describes the analysis of the COVID-19 test results.</div><div class = "text-block"><span style = "font-weight:bold;">Principles of Examination</span></div><div class = "text-block">The kit is a qualitative in vitro nucleic acid amplification assay to detect the new coronavirus 2019-nCoV identified in China in 2019 using Reverse transcription PCR of RNA derived from specimen of throat swabs and Bronchoalveolar Lavage Fluid (BALF) from individuals suspected to have been exposed to the virus. </div><div class = "text-block">The kit is based on in vitro RT-PCR with fluorescent probes. Primers and sequence-specific fluorescence probes were designed tailored to the high conservative ORF1ab region of the 2019-nCoV genome. The probes are oligonucleotides with attached fluorophores at the 5' end (FAM as reporter) and at the 3’ end (quencher). In addition, a specific primer and probe set designed to human b-actin is included as an internal reference with fluorophores VIC/HEX attached at 5’ end as reporter. During the PCR procedure, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye when the probes hybridize to the target DNA. 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 Real Time allows the qualitative detection of 2019-nCoV in specimens.</div><div class = "text-block">Analysis of the COVID19 test results will allow the qualitative detection of the 2019-nCoV nucleic acid in specimens from patients with suspected 2019n-nCoV. This process will enable the assessment of the infection in clinical and public health practice. The workflow for reporting results from this test is illustrated below:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/1618c1a37f153002e3885d7ebd72cd0a4e653895bbeb80a8934c28142d836cb2/b6gd7vcw.png" /></div></div>

[STEPS]
?. [RNA Transfer: Biomek FX Setup]
Open the Biomek software, and select the program “qPCR Setup” which is located in the “Covid-19” project:
Note: The RNA transfer into PCR plate  can also be done manually with a multichannel pipette with filtered tips in a prepared PCR Hood (10 μl of RNA from RNA plate into corresponding wells of the PCR plate, 10 μl of positive control into empty well H12).
?. [RNA Transfer: Biomek FX Setup]
Click on “Instrument Setup” as shown below to view the deck layout:
?. [RNA Transfer: Biomek FX Setup]
Wipe the deck using an RNaseZap wipe, followed by an alcohol wipe or .
[ethanol]
?. [RNA Transfer: Biomek FX Setup]
Place 2 boxes (lid off) of 50 μl filter tips as shown above.
?. [RNA Transfer: Biomek FX Setup]
Take the RNA sample plate (Eppendorf LoBind 96-well skirted plate) with the SPL barcode on the front side, and place in position P16.
?. [RNA Transfer: Biomek FX Setup]
Pulse spin the ABI MicroAmp Optical plate containing the qPCR reagents mix in the table top centrifuge if it has been stored at , then stack on a black Greiner CellStar plate. Place on the deck in position P17.
4 °C
?. [RNA Transfer: Biomek FX Setup]
Make sure you scan the barcode of the qPCR plate and the RNA sample plate on the Clarity LIMS. This will link the barcode of the qPCR plate with the RNA samples.
?. [RNA Transfer: Biomek FX Setup]
To START the program press the Run button (green triangle) located in the software.
The robot carries out the following automated protocol:1.    Load 50mL tips using pod 1.2.    Aspirate 10mL of RNA samples (10mL/sec).3.    Dispense 10mL to the qPCR plate (10mL/sec).4.    Unload tips.5.    Load one 50mL tip using pod 2.6.    Aspirate 10mL of TET buffer from position A1 of the microfuge rack.7.    Dispense 10mL to the qPCR plate position G12 (this constitutes the ‘negative control’).8.    Unload tip.9.    Load one 50mL tip using pod 2.10.  Aspirate 10mL of Positive Control from position D1 of the microfuge rack (20mL/sec).11.  Dispense 10mL to the qPCR plate position H12 (30mL/sec).12.  Unload tip.
?. [RNA Transfer: Biomek FX Setup]
The prompt on the screen will remind you to remove two tips from position G12 and H12 of the "50 μl Tips 1" box. Click OK to continue.
?. [RNA Transfer: Biomek FX Setup]
The next prompt will remind you that the MicroAmp plate should be stacked on top of a black Greiner plate.
?. [RNA Transfer: Biomek FX Setup]
Home the instrument by going to “Instrument” and selecting “Home All Axes”.
?. [RNA Transfer: Biomek FX Setup]
Seal the RNA sample plate using a Thermo Fisher PCR seal and move to storage at  .
-80 °C
?. [RNA Transfer: Biomek FX Setup]
Handover both  MicroAmp plate to RT-PCR operator or store temporarily at .
4 °C
?. [RT-PCR Set Up]
Take PCR Plate to PCR lab. On designated laptop in PCR lab, open "plate handover" App in Chrome browser.
PCR machines are situated in a separate area of the building to minimise risk of contamination of pre-PCR steps
?. [RT-PCR Set Up]
To confirm receipt of plate, click in empty field box in app, then scan the barcode with a handheld barcode scanner to enter PCR plate name. Hit SUBMIT.
?. [RT-PCR Set Up]
Check seal integrity on PCR plate and spin briefly in underbench centrifuge ().Open the sample drawer of an available QuantStudio 3 PCR machine and place PCR plate.
Centrifuge: 1000 33, 1 min
?. [RT-PCR Set Up]
Confirm that the barcode is visible and facing the front of the machine, and A1 is on top-left side.
?. [RT-PCR Set Up]
On the PCR machine-associated laptop, open the "design file" app in Chrome browser.
?. [RT-PCR Set Up]
Click in empty field box in app and scan the barcode on the PCR plate with handheld barcode scanner to enter PCR plate name. Hit SUBMIT.
?. [RT-PCR Set Up]
Download of text file will happen automatically. Save to “Plate Layout” folder on Desktop with the Barcode as filename.
?. [RT-PCR Set Up]
At this point, a second operator must confirm plate orientation.
?. [RT-PCR Set Up]
Open QuantStudio software.
?. [RT-PCR Set Up]
Click on the button to “Open Existing Experiment” and find the Covid-19 Mater Template.edt on Desktop.
?. [RT-PCR Set Up]
Click in field box for Barcode and use handheld scanner to scan plate barcode again to populate the field and replace default.
?. [RT-PCR Set Up]
Click in field box for Name and scan barcode again to replace default, which is “covid 19 master template”.
?. [RT-PCR Set Up]
Click in field box for User and enter your Crick user name.
?. [RT-PCR Set Up]
Under “File” menu click on “Import Plate Setup” and browse to identify the text file with same barcode on Desktop\Plate Layout. Hit “Apply”.
?. [RT-PCR Set Up]
You should be prompted that current info will be lost and replaced. Hit “yes”.
?. [RT-PCR Set Up]
Confirm the following settings by clicking on “Method” on menu near top of window.  ABCDE1StepCycleTemperatureDurationFluorescence measure211 cycle50°C20 minutesN321 cycle95°C10 minutesN4340 cycles95°C15 secN560°C30 secY
ABCDE1StepCycleTemperatureDurationFluorescence measure211 cycle50°C20 minutesN321 cycle95°C10 minutesN4340 cycles95°C15 secN560°C30 secY
?. [RT-PCR Set Up]
When prompted, click on “Edit” and enter password: covid192020.
?. [RT-PCR Set Up]
Confirm following settings by clicking on “Plate”.(Advanced Setup Tab) Target1: FAM, Quencher: None for Covid-19(Advanced Setup Tab) Target2: VIC, Quencher: None for internal control(Quick Setup Tab) Passive Reference Dye: None in “Plate” window in Quick Setup Tab that plate is populated with sample barcodes.
?. [RT-PCR Set Up]
Click on “Run” window, and hit “Start Run” button.
?. [RT-PCR Set Up]
When prompted, save barcode.eds file in “Covid19 Results” folder on Desktop.
?. [RT-PCR Set Up]
FINAL CHECKPOINT (stop the run if the following is not true): The PCR run should last .
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
The run file from the analyser (EDS file) are exported to a shared drive. This folder will be called “Primary Review”.
IMPORTANT: The file must have a unique name that identifies the run and can be linked to the electronic sign-off in Clarity. In this case it will be the unique barcode associated with the patient hospital number.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
First reporter/checker should: Access the run in Quantstudio 1.4.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Ensure threshold is set to automatic.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Check the run as a whole on the amplification plot.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
If the run shows a lack of typical PCR amplification across the plate (whole plate failure), reject the run and repeat set up.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Ensure positive control is ‘positive’ for nCOVID19 and internal control and the negative controls are ‘negative’ for nCOVID19 and internal control:Blank control (TET Buffer) and extraction control: Ct values at FAM and VIC/HEX channels are greater than 37 and 35 respectively or undetermined.Positive control: Standard curves at channel FAM and VIC/HEX channels are showexponential amplificationwith Ct values not greater than37 and 35 respectively.
Above requirements should be met on the individual plate otherwise the entire plate is invalid. However in borderline cases this may be affected by manual thresholding and can be refered to the second reporter for final decision on plate failure.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Use the text box to the right of the properties tab to include any additional information that needs to be communicated to the second reporter.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Click on properties tab and put their name in the “user” free text box.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
In the results tab check all samples using the algorithm below and for cases which should be omitted right click the well and select OMIT.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
The checked .eds file is then saved to the /report1 folder from where it will be uploaded to the online portal.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
The first reporter must extract the plate geneology data from ClarityLims allowing them to documentthe history of a plate so the stages and operators involved. This will support their sign off decision.A failed genealogy request will be illustrated as: A successful genealogy request will be illustrated as:
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
If entire plate has failed and needs to be retested, go back to stored RNA and repeat RT-PCR.
?. [Second Reporter QuantStudio 3 Analysis:  Recording, Calculation of Results and Laboratory Clinical Interpretation]
The second reportor will be able to download the run results for each run and its duplicate through a web app. Following this they will analyse each plate individually and upload the analysed results back onto to the web app and a final outcome for each sample will be determined based on the result from both runs.Positive and negative results will all be automatically generated in the HSL LIMS system (WinPath) based on Cts and concordance.
?. [Second Reporter QuantStudio 3 Analysis:  Recording, Calculation of Results and Laboratory Clinical Interpretation]
The second reporter repeats the checking of positive and negative controls as described above () and makes sure of the following:The acceptance criteria have been passedThe overall plate curves suggest appropriate amplificationThere is a complete audit trail for all sample processing steps (At the time of writing this functionality has yet to be completed but will be available in a later edition)
?. [Second Reporter: Import of eds file]
The results are checked by the second reporter using the online portal.Any completed plate runs will have the status “Ready to validate” which is a clickable link. The extraction container relates to the plate of RNA that each run originated from, duplicates can be identified as they have the same extraction container ID. The two runs should be opened at the same time thus locking each from interaction by another reporter.
?. [Second Reporter: Import of eds file]
When the “Ready to validate” link is clicked the run will become locked to other reporters for an hour. If more time is need, the reporter can click “Get more time” or if they do not continue the validation the can click “Release the lock” to allow other reporters to have access to these runs. To import the eds file the reporter can click on “Download report file”.
?. [Second Reporter: Using QuantStudio Software]
The QuantStudio software will open and the password for the locked template is covid192020 - this must be entered to allow for customisation of the eds file.Check the comments box under the properties tab for any comments made by the first reporter.The eds file automatically opens to the results page in log format. If the plate to be analysed is only half full, to remove any of the wells that are not to be analysed go to the plate screen and select these wells and deselect the targets.
?. [Second Reporter: Using QuantStudio Software]
Curve visualistion: To visualise S curves back on the results page change to a linear graph by clicking the eye symbol and change this under graph type. Then press ANALYSE button, top right.Covid-19 fluoresces in the FAM channel and is shown in red. The internal control fluoresces in the VIC channel and is shown in blue. To visualise the curves for individual samples click on their well, e.g. the positive control (left) and negative control (right) shown below.To look closer at any curves or identify which well they are in you can drag the mouse over these to select them and they will become highlighted and the wells they correspond to will be marked with a dotted square box.
?. [Second Reporter: Using QuantStudio Software]
Targets: To visualise just one target at a time, either covid-19 or internal, this can be done under the plot settings (eye symbol) by changing the target from all to covid-19 or internal.
?. [Second Reporter: Using QuantStudio Software]
Ct visualisation: For an easier view of the Ct produced by each curve use the list graph list screen rather than the 96 well screen on the right hand side (button and Ct column circled).
?. [Second Reporter: Threshold]
Manual baseline and threshold:The baseline will be automatically set between 3 to 15 cycles as according to the BGI kit guidelines however may be manually adjusted to reflect the amplification on the plate. Click on the eye symbol and untick the auto baseline box (circled below left) then drag the baseline end to just before the first true amplification on the plate. The Ct values will automatically be reanalysed once this has been moved. This is best done in the linear graph.Once the baseline has been adjusted the result analysis can be done using manual thresholding to remove any noise or abnormal curves that could be interfering with the appropriate Ct being given for each sample. In some cases the automatic thresholding will have called all results appropriately and the threshold can be manually set to the same level.Click the eye symbol and untick the auto threshold box, (circled below left). The threshold line will then become draggable by mouse (below right) and can be raised to a level above any “noise” on the plate. The Ct values will automatically be reanalysed once this has been moved. This is best done in the log scale graph.
?. [Second Reporter: Result Anaylsis]
The second reporter should again check that the controls meet these criteria:Positive control:Standard curves at channel FAM (Covid-19) and VIC/HEX (Internal Control) channels showexponential amplificationwith Ct values not greater than 37 and 35 respectively.Negative controls: Ct values at FAM and VIC/HEX channels are greater than 37.0 and 35 respectively or no data available. There are two negative controls – one with viral inactivation L6 buffer that has been through the inactivation process (well F12) and one no template control at the RT-PCR stage (well G12).
?. [Second Reporter: Result Anaylsis]
The three following results are the possible outcomes for each sample:  ABC1Covid-19Internal Control2PositiveCt Ct 3NegativeCt >37.0 or undetermined,
Any non exponential amplificationCt 4Sample failureAny Ct, any non exponential amplificationUndetermined or >355Sample failure (2)Non exponential amplification of both or either target with Ct values Non exponential amplification of both or either target with Ct values Positive and negative results are generated automatically in the LIMS based on the Ct values in the uploaded xls and released immediately. Sample failure results are generated automatically in the LIMS by an “Invalid” comment in the xls results and also released immediately.
ABC1Covid-19Internal Control2PositiveCt Ct 3NegativeCt >37.0 or undetermined,
Any non exponential amplificationCt 4Sample failureAny Ct, any non exponential amplificationUndetermined or >355Sample failure (2)Non exponential amplification of both or either target with Ct values Non exponential amplification of both or either target with Ct values
?. [Second Reporter: Result Anaylsis]
Plate failure: The plate should be failed if there is signal of
?. [Second Reporter: Result Anaylsis]
Exporting the xls file from QuantStudioOnce the results have been analysed and the plate has passed the eds file must be exported to an xls file to upload back onto the results portal. Under the export screen select the browser location to export this file to, ensure this is a .xls file (not .xlsx) and then click export button circled. Once exported the xls form will automatically open, save in a folder on your drive. The modified eds file must also be saved in this folder.
?. [Second Reporter: Result Anaylsis]
Uploading the xls file to the online portalFor each run upload the .xls file (not .xlsx) to the portal by clicking browse and uploading the file. Do the same for each with the eds file. Once both files have been uploaded press the “Validate” button. The reporter will then be returned to the Referrals list where any other reports awaiting validation will be available.
?. [Sample Requeue Process ]
If the PCR plate has failed for one run, the RNA from this run will go into a new barcoded PCR plate.The RNA team will requeue the RNA extraction plate into the aliquot step and scan the new destination plate barcode into the Claritylims system.This will maintain the link to the original barcoded RNA plate and samples, so that we can also identify the number of times this plate has been repeated.
?. [Result File Structure]
The result file is the Excel output from the QuantStudio. It has 3 tabs:  ABC1Sheet #Sheet NameDescription21Sample SetupInformation about the specimens contained in each well, and the run setup.32Amplification DataRaw data of the well curve X/Y coordinates.43ResultsThe main sheet containing the well numbers, sample numbers and result values (CT values). This will be used for import to Winpath.The results tab has a table starting on row 51 with the results. The key columns for import are:  ABC1Column positionColumn headerDescription2DSample NameThe lab number associated with the sample.3ETarget NameThe assay processed. This needs to be mapped to the result code in Winpath as part of the interface.4ICTThe quantitative result. This will either be a number or ‘Undetermined’. This will interface to an internal line in Winpath. Winpath rules will then generate the final result.Example:Translates to this result in Winpath:
ABC1Sheet #Sheet NameDescription21Sample SetupInformation about the specimens contained in each well, and the run setup.32Amplification DataRaw data of the well curve X/Y coordinates.43ResultsThe main sheet containing the well numbers, sample numbers and result values (CT values). This will be used for import to Winpath.
ABC1Column positionColumn headerDescription2DSample NameThe lab number associated with the sample.3ETarget NameThe assay processed. This needs to be mapped to the result code in Winpath as part of the interface.4ICTThe quantitative result. This will either be a number or ‘Undetermined’. This will interface to an internal line in Winpath. Winpath rules will then generate the final result.
The files should be dropped to the sFTP server RESULTS folder.The files should have a unique name.If a run is exported with failed run controls by mistake the interface will prevent it being transmitted to Winpath.The Winpath rules will look for technical failures and prevent reporting if an invalid result is identified.
?. [Result File Structure]
SFTP ServerThe SFTP server is hosted by HSL.Folder structure:
?. [Preparation of PCR master mix plate]
Take required number of BGI RT-PCR kits from   freezer (2 Kits per 96w plate of RT-PCR reactions, 4 kits per full RNA plate since RT-PCR reactions are run in duplicates) and remove the 2019-nCoV Enzyme mix to maintain , remove the Negative Control to   freezer, and allow the remaining tubes (2019-nCoV Reaction mix, Positive Control) thaw up at .
Reagents are stored at -20ºC. Take out all the kit contents and thaw them thoroughly at ambient temperature. Vortex and centrifuge briefly. The enzyme mix should be kept on ice at all times. RT-PCR mastermix is aliquoted into PCR plates in designated hoods in the HTS tissue culture room. All personnel must have full PPE when entering the tissue culture room, which includes clean lab coat, fresh nitrile gloves, and full-face guard. Additionally, the individual pipetting in the hood must wear disposable forearm sleeves.
-20 °C
on ice
-20 °C
-80 Room temperature
?. [Preparation of PCR master mix plate]
2019-nCoV Reaction mix has thawed, retrieve 2019-nCoV Enzyme mix tubes from ice, quick spin down all tubes.
?. [Preparation of PCR master mix plate]
Briefly spin the plate at , and store  or at , together with the thawed positive control tube.
Centrifuge: 1000 33, 1 min
on ice
4 °C
?. [RT-PCR Set Up]
After the amplification is complete, remove PCR plates from the thermal cycler and discard plate for autoclave and decontamination.
?. [RNA Transfer: Biomek FX Setup]
Place a black microfuge tube rack in position P19.
?. [RNA Transfer: Biomek FX Setup]
Place an eppendorf tube (cap open) with TET buffer in position A1and the Positive Control tube (cap off) in position D1of the microfuge tube rack.
?. [RNA Transfer: Biomek FX Setup]
Make sure to purge the Span-8 syringes until no bubbles are seen in the tubing.
?. [RNA Transfer: Biomek FX Setup]
Check the level of the Biomek FX Span-8 water container. Re-fill with Milli-Q water if the level is too low. Empty the waste bottles in the sink when they start getting full.
?. [RNA Transfer: Biomek FX Setup]
The last prompt will remind you of the microfuge rack setup:    A1: TET buffer in Eppendorf tube    D1: Positive Control
?. [RNA Transfer: Biomek FX Setup]
Once the program is complete, carefully remove the MicroAmp plate from the deck.
?. [RNA Transfer: Biomek FX Setup]
Seal with a MicroAmp Optical Adhesive Film.
?. [RNA Transfer: Biomek FX Setup]
Press the seal down to ensure a good seal on each well using the MicroAmp Adhesive Film Applicator.
?. [RNA Transfer: Biomek FX Setup]
Repeat steps  to  with a second MicroAmp plate for a total of 2 transfers.
?. [Preparation of PCR master mix plate]
Clean hood and everything that enters hood with DNA ZAP by spraying Solution 1 followed by Solution 2 and then wiping dry with clean paper towels.
?. [Preparation of PCR master mix plate]
Dispense PCR Master mix into plates as follows:- For half plate, leave PCR mix in tube and use an electronic single channel pipette fitted with a filter tip to draw up 100 or 200µl and repeatedly dispense 20µl into individual wells of the 96 well PCR plate.- For Full Plate or more: Using an L-1000 pipette with filtered tip from Blue tip box, pipette all the Master Mix from individual tubes into a 25ml individually wrapped reservoir. Dispense 20ul of Master mix into all wells of a barcoded MicroAmp™ Fast optical 96-well plate, using an LTS 20-300µl multichannel pipette
Pipette settings: From Main menu, select Multi-Disp setting with Aliquot volume 20 µl, 8 aliquots, 1/1 Asp/Dsp Speed
?. [Preparation of PCR master mix plate]
Eject any remaining PCR master mix left in tips back into reservoir.
?. [Preparation of PCR master mix plate]
Seal the plates with AlumaSeal. Check each well is sealed.
?. [Preparation of PCR master mix plate]
Transfer remaining PCR master mix back into a Reaction Mix tube, label with date, and put in box in fridge under the bench.
?. [Preparation of PCR master mix plate]
Clean up hood by spraying with both DNA ZAP solution 1 followed by DNA ZAP solution 2 and wiping with paper towel.
?. [First Reporter QuantStudio 3 Analysis: Initial Data Analysis, Internal Quality Control Procedures and Criteria]
Each run will be run in duplicate and once both runs have been checked and passed the .eds file for one of these runs (“principal run”) is then saved to the /report1 folder from where it will be uploaded to the online portal.
?. [Second Reporter: Threshold]
Adding CommentsINVALIDIf a sample hasnon exponential amplification (red arrow below) of both or either target with Ct values
?. [Second Reporter: Result Anaylsis]
Duplicate Run OutcomeThe second reporter will have completed the steps outlined in 6.3 in QuantStudio for both runs and applied the same acceptance criteria for the controls and patient samples outlined in 6.4.The automated outcome will be calculated by the logic in the online system that will take into account the Ct value and any well comments. Once the results for both runs have been uploaded the following outcomes will be possible:  ABCD1NoCovid CT Value 1Covid CT Value 2Expected result21POSITIVE32>37/UD>37/UDNOT detected4335-37>37/UDNOT detected54>37/UD35-37NOT detected65>37/UDInvalid (discordant)76>37/UDInvalid (discordant)Any samples with the comment invalid in either one or both of the runs will be called invalid.Any samples that have an internal control failure in one or both runs will be failed.
ABCD1NoCovid CT Value 1Covid CT Value 2Expected result21POSITIVE32>37/UD>37/UDNOT detected4335-37>37/UDNOT detected54>37/UD35-37NOT detected65>37/UDInvalid (discordant)76>37/UDInvalid (discordant)