detection of 2 sars-cov2 using the abi 7500 fast
TRANSCRIPT
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Public Health Wales Microbiology Cardiff Molecular Virology
Detection of 2 SARS-CoV2 using the
ABI 7500 Fast Document and Version: CDVSOP 201.2
Author: R.Rees/C. Moore Authorised by: L Chichester
Date authorised: 13 March 2020
Publication/ Distribution:
1. NHS Wales (Intranet)
Review Date: 13 March 2023
Purpose and Summary of Document:
Document to provide guidance to perform SARS-CoV2 screening and confirmation.
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Document amendments by completing the following table:
Date Section
No. Page No.
Version No.
Description
25/02/20202 All All 2
Amendment of viral
nomenclature. Alterations made to the
guidance document to reflect changes in
process.
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Contents
PURPOSE OF THE EXAMINATION 4
PRINCIPLE AND METHOD 4
CROSS REFERENCES 4
HEALTH AND SAFETY 5
SPECIMEN COLLECTION AND TRANSPORT 5
SPECIMEN TYPE AND TEST SELECTION 5
EQUIPMENT AND REAGENTS 6
SPECIMEN PROCESSING 7
QUALITY CONTROL PROCEDURES 13
INTERFERENCES AND LIMITATIONS 14
RESULTS REPORTING 14
AUTHORISATION 17
REPORT ISSUE (Interim/Final/Additional) 17
REPORTING TO OTHER DEPARTMENTS 17
REFERRAL TO REFERENCE UNITS 17
Appendix 1 17
Appendix 2 Guidance for SARS-CoV2 Confirmatory Testing 21
Appendix 3 SARS-CoV2 Testing Record 24
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1. PURPOSE OF THE EXAMINATION
This document outlines the process for detecting the envelope (E) gene of
SARS-CoV2 from clinical samples collected from patients with suspected infection. This assay is designed to screen for all coronaviruses of the Asian
lineage of beta coronaviruses that include the bat SARS coronaviruses as well as the SARS-CoV2. The assay has been shown to have good sensitivity
and specificity for this group of viruses with no cross reactivity shown to human coronaviruses (229E, OC43, NL63 and HKU1) or other respiratory
viruses or bacteria.
2. PRINCIPLE AND METHOD
Clinical samples received for testing in the laboratory, are processed under BSL3 facilities and pre-treated with a lysis buffer containing guanidinium
thiocyanate that acts by disrupting all cellular, bacterial and viral surface proteins to release nucleic acid into solution. At this point the sample is
rendered ‘safe’ (not infectious) and can be transferred to a BSL2 laboratory for total nucleic acid purification using an automated extraction system.
Following nucleic acid purification, amplification and simultaneous detection of the target gene is undertaken using a set of optimised primers, probes
and enzymes (including reverse transcription) in a real-time system.
The assay is qualitative, in that the results are reported as detected based on a threshold cycle (Ct value) of <40; or not detected based on a Ct value
of >40. The positive control for the reaction is an RNA transcript of the E gene
(European Virus Archive) at two concentrations representing a viral load of approximately 100 RNA copies/µl and 10 copies/µl. These dilutions are
performed in duplicate and recorded after each assay run to monitor assay integrity over time and batch.
3. CROSS REFERENCES
CDMSOP 020 EASYMAG Total Nucleic Acid Extraction CDVLSOP 002 TrakCare LIMS Patient and Specimen Data Entry
CDVLSOP 003 TrakCare LIMS Result Entry CDVLSOP 007 TrakCare LIMS Use of Worksheets
CDVLSOP 014 Using the TrakCare Storage Module in Virology CDHS 014 Discard & Disposal Process
CDHS 015 Guidance for Staff working in Containment Level 3 MDHST 017 Competency programme for working in Containment
Level 3 WVSOP 001 Virology Test Selection
Refer to section 4.0 Health & Safety for associated risk assessments.
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4. HEALTH AND SAFETY
This method should be carried out using principles of good laboratory
practice at containment levels 3. All risk and COSHH assessments must be read prior to use of method. Personal protective equipment (PPE), such as
gloves and goggles, MUST be worn where indicated.
Risk Assessment MDRA 001 Whole Workplace Risk Assessment
MDRA 005 Use of Equipment MDSTRA 002 Swab Samples
MDSTRA 004 Risk Assessment for Processing Respiratory Samples MDHSGUID 011 Guidance on Transportation and Packaging of Enhanced
Cat B Specimen
COSHH Assessment
MDCOSHH 047 Sodium hypochlorite (Bleach) MDCOSHH 150 Guanidine thiocyanate
MDCOSHH 350 RNase Wipes MDCOSHH 439 Magnetic silica
MDCOSHH 519 Chemgene
5. SPECIMEN COLLECTION AND TRANSPORT
Transport
Samples from other laboratories and the community are transported to the
laboratory via a courier. Samples and waste are stored in a designated SARS-CoV2 area within Virology Specimen Reception prior to processing.
Transportation of samples from within UHW is co-ordinated by the medical
team and designated BMS performing SARS-CoV2 testing.
2 x Throat swabs are expected from any patient being tested for SARS-CoV2
Refer to Appendix 1 for further information.
6. SPECIMEN TYPE AND TEST SELECTION
One throat swab is to be tested for the SARS-CoV2 target gene in the E
gene screening assay. Code all request forms with a RESPL test set and
sample type. All requested received must be assigned to outbreak number
98 on data input.
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Throat swabs are the preferred sample for routine testing. The virology
consultant may request testing on additional samples for specific cases. This
will be reviewed by the virology senior management team the following day
to assess if any permanent changes are needed to the SOP.
ITU Surveillance and GP Surveillance requests will require a full respiratory
screen in addition to SARS-CoV2 testing, an additional RESPL test must be added to report the Biofire/ Lumienx result. Note;- Requests from other
hospitals may have already reported Biofire results, in this instances further
testing at UHW is not required.
7. EQUIPMENT AND REAGENTS
7.1 EQUIPMENT
Equipment Manufacturer Location Use
Microcentrifuge Eppendorf Room 147 Sample
Preparation
Automated multi-channel pipette
Biohit (Biomerieux)
Room 137 Extraction set-up
EasyMag Biomerieux Room 137 Extraction
ABI 7500 Fast Applied
Biosystems Room 134
Amplification & detection
7.2 REAGENTS
Lysis Buffer, 2ml bulk stock and small 0.9ml located in the Clean Room (132). In-use 2ml lysis buffer and small 0.9ml lysis buffer is located in the
designated SARS-CoV2 area within Room 147.
The reagent stock for amplification and detection (to include Primer probes and ABI FAST mix) are kept at -20C in room 132. The probe mix is
aliquotted into volumes for multiples of 10 reactions. In-use lots are stored in a labelled box at -20C in room 137. Aliquots of molecular grade water
are kept in a labelled box in the Williams refrigerator.
The positive control aliquots are stored in the freezer in room 137. Check
labelling on the freezer door for exact location within the freezer.
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8. SPECIMEN PROCESSING
8.1 SAMPLE PREPARATION: Routine Screening
1. Receive samples into designated area within specimen reception 2. Transport samples to Virus Isolation to begin processing
3. Unpack samples out of cardboard box on the bench as per normal sample practice
4. Within the class 1 cabinet unpack sample from plastic container, It should still have a minimum of 2 barriers to the potentially
infectious material. i.e. the sample bag and primary container. 5. Match sample demographics with the request form.
6. Ensure request forms are date stamped 7. Label the request form, 2 small lysis, 1 large lysis and 1 skirted 2 ml
extract tube for every sample type that requires extraction. 8. Number both throat swabs, 1x dry throat swab (swab a) for SARS-
CoV2 testing must be broken into a corresponding numbered small
lysis buffer and incubated for a minimum of 10 minutes.
9. The second swab (Swab b) must be broken into its corresponding
numbered small lysis. Label the lid with an S and store in the
designated rack labelled secondary tube.
10. Swab A is to be stored in the virology respiratory original storage
after sample preparation is complete
11. Swab b can be discarded after validation of a negative SARS-CoV2.
12. In the event that swab A is positive swab b is to be referred along
with the extract to genomics once confirmation testing, if applicable,
has been performed.
13. Request forms are to be directed to a designated staff member for
data input and population of the laboratory list.
14. Discard boxes as normal process. (Hays DX to be returned in red
sacs for cleansing and reuse)
15. All waste generated must be disposed of in orange bags. After
validation of a negative SARS-CoV2 result sweetie jars of snapped
swabs can be disposed of as normal.
8.2 SAMPLE PREPARATION:- Known Positives (Cat3)
1. Transport specimen + labelled small lysis to containment level 3
within a sample transport box
2. Samples processing must be performed within containment level 3
3. Door code – Bottom- middle-top
4. Ensure all 3 hoods are switched on to maintain appropriate
negative pressure
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5. Ensure the cat 3 door is shut
6. Ensure containment level 3 protocol is followed and appropriate PPE,
all samples must be processed within the hood.
7. Snap/Cut swab into 0.9ml lysis buffer & vortex well (ensure swab is
snapped/cut off as short as possible to allow space for pipette tip to
reach liquid).
8. Once the samples have been incubated for 10 minutes they are safe
to remove from containment level 3 for further processing.
9. Before leaving cat 3:-
a. Wipe down the outside of the lysis buffers with disinfectant and
place in a plastic bag. Wipe down the plastic bag with
disinfectant before you leave
b. Spray the sample rack with ethanol spray
10. Place the sample rack, plastic bag containing small lysis and
remaining episode numbers into the sample transport box
11. Transport sample to the isolation laboratory
12. Place sample rack into the bleach bucket immediately
13. Label 1 large lysis and 1 skirted 2 ml extract tube for every sample
type that requires extraction. Ensure sample type is specified on
both the large lysis and extraction tube
8.3 SAMPLE EXTRACTION: Performed on the E Mag and Easy
Mag
SAMPLE EXTRACTION (Room 137)
1. DO NOT EXTRACT POSITIVE CONTROLS
2. Extract one extraction control (lysis buffer)
3. Centrifuge all sample and control lysis tubes for 2 minutes.
4. Transfer 250 µl of sample in 0.9ml lysis buffer to the corresponding
2ml lysis buffer tube. Vortex and centrifuge for 2 minutes.
5. Swab A is to be stored in the virology respiratory original storage
after sample preparation is complete
6. Log on to the extractor - the usernames and passwords are noted on
stickers on the monitor of each machine.
7. Start a new worklist on the extractor (see CDMSOP 020 for detailed
instructions). Enter requests as Generic 2.0.1, sample volume
0.200ml and extraction volume 110µl. Enter matrix as Other.
Record the lot number for the 2ml lysis buffer in the ‘lot number’ field.
8. Scan barcodes of all samples and controls. A prompt will occur if
inputting samples/control barcodes that have been previously
extracted - click yes to create a duplicate.
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9. To manually enter episode numbers/controls, select the sun icon for
a new entry and type in the sample or control ID.
10. Create a new extraction worksheet on the next screen by selecting
the sun icon. Name the worksheet COVID followed by the run number
and date (dd/mm/yy). Using the arrows import all samples/ controls
to the worksheet.
11. Equipment & Reagents for extraction (all stored in room 137):
Magnetic Silica – stored in Williams refrigerator.
Extraction Buffer 3 – decanted weekly (Monday morning) into
universal container and stored in cupboard under EasyMag 2.
EasyMag Cartridges and Tips - stored in cupboard under EasyMag
2.
Microwells & holders – stored in cupboard under EasyMag 2.
Ms2- Located in the Luminex box
12. Place required number of EasyMag cartridges into dedicated metal
rack. Cartridge Tips can be preloaded into the EasyMag at this point.
13. Label cartridge positions with the corresponding episode number/
control/ following the order on the EasyMag screen.
14. Place the required number of microwells into a holder. One strip is
required per EasyMag cartridge.
15. In a safety cabinet add 10µl of MS2 (NxTAG Internal Control) to each
well of the EasyMag cartridge. Ensure that the MS2 is dispensed at
bottom, narrow part of the well. MS2 should only be added to patient
specimens and the negative control.
16. Transfer the entire lysis buffer containing sample/control to the
corresponding labelled well in the cartridge using disposable fine tip
pipettes. Keep bubbles to a minimum.
17. Remove the required number of silica tubes from the Williams
refrigerator. Each silica tube (1.2ml) contains enough for two
cartridges (1x 600µl silica aliquot per 8 samples). Vortex the 1.2ml
silica tube and aliquot 600µl of silica from the original tube to a 1.5ml
Sarstedt tube.
18. Set the automated pipette to programme 1 by pressing SELECT to
change the programme number, followed by ENTER. To operate the
pipette, press the central dark blue button to aspirate 550µl of buffer
3 and again to dispense the liquid into each 600µl aliquot of silica. To
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eject the tip, press one of the small buttons on either side. Vortex the
silica/buffer 3 solution.
19. Change the pipette to programme 2 to aspirate the silica, then dispense
125µl into each of the microwells (eject the first volume and any extra
silica back into the silica tube).
20. Set the pipette to programme 3 and select the required number of tips.
Aspirate 100µl of the silica from the microwells and transfer into the
EasyMag cartridge. The pipette will automatically mix the content of
the cartridge three times. Repeat this step for all EasyMag cartridges,
changing tips for each cartridge.
21. Place the EasyMag cartridges on the EasyMag. Barcode in the cartridge
positions, cartridge ID, elution buffer (buffer 3) and silica lot numbers.
22. Begin the run by pressing the PLAY button (green triangle) on the right
hand side of the screen.
23. A full extraction takes approximately 40 minutes.
24. At the end of the run, check for any errors on screen and note in green
maintenance file.
25. The PDF of each run is now saved as part of the Molecular Laboratory
monthly checklist. These files are transferred to the G drive
(G:\VIROLOGY\Virology Molecular Testing\Respiratory
PCR\LUMINEX\EasyMag Extraction Reports).
26. The extracts must be transferred to the corresponding labelled extract
tube within 30 minutes of the extraction finishing.
27. Centrifuge the extract tubes for 30 seconds.
28. The extracts can be kept at 4°C until testing. Extracts must be taken
back to room 147 after testing is complete.
8.4 PRE AMPLIFICATION:
1. Record sample numbers on the SARS-CoV2 worksheet in the order in
which the controls and specimens are to be tested. This can be completed by hand or electronically. Episode numbers are to be
written in full. 2. Testing :-
i. Patient samples singularly ii. followed by the negative extraction control in duplicate
iii. followed by positive control (10-3 & 10-4) in duplicate iv. Followed by a water
3. All mastermixes should be prepared in the clean area of Room 137 - only use YELLOW racks in the set-up area.
4. All reagents required should be removed from the freezer and allowed to defrost completely before use. Do not mix batches of reagent.
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5. Record all reagent batch numbers and expiry dates on the worksheets
as required. 6. All reagents should be mixed and centrifuged before use. The ABI
Virus Fast Mix is very viscous and needs to be well mixed by inverting several times before centrifuging.
7. Add ABI Fast Mix to the primer probe aliquots as per quantities on the
worksheet. 8. Add molecular grade water to the mix as per quantities on the
worksheet. Multiple aliquots should be combined. 9. Gently mix and pulse centrifuge constructed mastermix.
10. Mastermixes should be used within 15 minutes of preparation and not left exposed to the light, due to possible degradation of the
fluorescent probes.
8.5 AMPLIFICATION SET-UP:
1. Place the appropriate number of 0.2 ABI Fast reaction strips in the plastic holders. 96 well PCR plates can also be used when required.
2. Add 20l of Sarbeco /RNaseP mastermix directly into the
tubes/well.
3. Add 5l of each sample or control extract to the appropriate
tubes/wells, as per the order on the worksheet.
4. Carefully cap each strip. Remember to place an orientation
mark/number on each strip. Check that all caps are securely sealed.
5. Transfer the 0.2ml tubes to room 134 in the plastic holder (take
worksheet with you).
Note: Remove laboratory coat and wash hands before leaving the
laboratory.
8.6 NUCLEIC ACID AMPLIFICATION AND DETECTION
If instrument is ready to use skip to step 4.
1 Switch on the PC connected to the ABI 7500 Fast. The password is the
same as the log-in name. Both are case sensitive. 2 Double click on the 7500 software icon and select OK to login as guest
when prompted. The system will now run through the calibration checks. 3 Select the Template icon from the front page (Figure1). A list of
templates will appear. Select the SARS-CoV2 testing template, the
template is available on ABI 1-3.
NOTE: Start up the ABI 7500 at least 15 minutes before use.
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Figure 1: Overview of the ABI 7500 screen software
4 Mix the tubes by gentle flicking and then using the small 0.2ml strip-tube centrifuge, centrifuge for 5 seconds.
5 Ensure there are no bubbles present in the tubes and no liquid on the lid re-centrifuge to disperse bubbles and no liquid is visible on the lid.
6 Carefully open the plate drawer of the 7500 fast by placing thumb into
the door indentation on the right hand side (Figure 2).
Figure 2: The ABI 7500 machine
7 Place strips into the appropriate ABI holder. A different holder is required if using a 96-well plate.
8 Place the strips in order, so the sample positions correspond to the worksheet. Place empty strips equally spaced across the holder to
balance the machine. 9 Carefully close the plate drawer by placing thumb into the door
indentation on the right hand side and applying pressure to the tray at an angle (Figure 3).
Figure 3: The ABI 7500 Fast plate drawer
10 Select Experiment Properties from the Setup menu on the left hand
side of the screen (Figure 4).
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Figure 4: Example Overview of the ABI 7500 software program
11 Enter the experiment name (SARS-CoV2 and date) into the Experiment
Name field and operator name into the User Name field.
12 Select Start Run. A prompt box will appear to save the experiment. Save in Experiments>Virology>2019 nCoV> Select Year> Select Month.
13 From the setup menu on the left hand side select Setup then select Plate Setup.
14 A new screen, with the tabs Define Targets and Samples will appear. 15 In the Define Samples field, assign the full episode and control name
to the designated sample box .Press the cursor after the addition of each sample number to move down to the next line.
16 Select the Assign Targets and Samples tab. 17 Assign the samples to the selected wells by selecting each well
individually and then assigning the correct episode number to the corresponding well by selecting the box next to the sample name.
18 Assign targets by highlighting all the wells and selecting SARS-CoV2 E gene +RNaseP
19 Select the Run menu on the left hand side to monitor the run.
20 Amplification and detection takes approximately 45 minutes.
9. QUALITY CONTROL PROCEDURES
A negative amplification control is extracted daily and tested on every run,
along with a water blank. The positive controls are tested daily. Single use aliquots can be found in a labelled rack in freezer. Positive controls do not
require extraction. The CT value of the positive control is plotted on Medlab
QC after each run before any results are released.
Expected CT Values 10-3 (31-33)
10-4 (35-37)
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Unusual results or trends must be discussed with the Senior BMS for the
section or the clinical scientists.
Positive patient samples (extract + swab in lysis) are to be referred to PENGU for sequencing. See Appendix 1 for further information.
Each specimen includes an internal control (RNAseP) to ensure there is sufficient cellular material for analysis.
10. INTERFERENCES AND LIMITATIONS
The assay has been validated to detect a lower limit of 1 RNA copy/µl of the
envelope RNA transcript with 100% detection of 10 copies/µl (represented
by the lowest transcript control). LLOD against SARS CoV is 10-3 RNA copies/µl and 100% detection at 10-1 copies/µl
Poor sample quality, the presence of PCR inhibitors (e.g. haem or high levels of protein) and/or virus present in the reaction below the 100% detection
limit may lead to results being reported as not detected.
11. RESULTS REPORTING
11.1 DATA ANALYSIS
1. Once the run has finished, data should be automatically analysed.
2. The assay should be checked initially on the multicomponent plot (third line down in the analysis tab). The blue lines relate to the FAM (SARS –
CoV2 E gene target) signal and brown lines relate to the NED (RNAseP target) signal and the red lines relate to the ROX reference dye signal.
3. Check the ROX (ABI reference dye) signals on the multi-component chart, the plots should have straight horizontal lines any deviation
should be noted. ROX should be tight with minimal spacing. 4. Then click onto the amplification plot at the top of the tab selecting ∆RN
vs cycle and select either linear for the full amplification curve or log to show the rate of amplification.
5. Check the thresholds for SARS-CoV2 E gene and RNaseP are correct, edit threshold if required. Thresholds should be set as : SARS-CoV2 E
gene (0.2) , RNaseP (0.1)
6. Check each well individually by target. Note any samples that cross the threshold (CT ≤40) for SARS-CoV2 Egene. Any detectable samples >35
CT must be repeated on the confirmatory platform, see Appendix 2. 7. All clinical samples should have an RNaseP signal. Make a note of
samples that have no RNaseP signal, RNaseP CT 35- 37.9, or RNaseP CT>37.9.
8. If any plots are not clear inform a Senior BMS. 9. Insert a pen drive into the USB port on the side of the laptop.
10. To export results select Print Report from the main menu at the top of
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the results page.
11. Select Experiment Summary, Results summary, Amplification Plot (∆Rn vs Cycle) and Results table (By well).
12. Save the experiment file onto the pen drive by selecting the Save icon from Print Preview.
13. Remove pen drive.
14. Remove all sample strips and dispose of into double gloves then into a sweetie jar. If a 96 well plate is used, dispose into a sweetie jar without
double gloving. 15. At the end of the working day turn off ABI 7500 Fast and exit programs
using either file exit or the exit cross at the top right hand side of the
main menu. Ensure all testing tubes are removed prior to shut down.
11.2 RESULT REPORTING
1. Transfer result report to the 2019 nCoV results folder. G drive:
Virology: Virology Molecular Testing: 2019 nCoV 2. Input SARS-CoV2 E gene positive control CT value into the Medlab
software, any result flagged in the red area must be brought to the attention of a senior member of staff.
3. All clinical samples should have a positive RNaseP signal with a CT of 37.9 or less. All negative samples with an RNaseP CT >37.9 should be
reported as insufficient. These results should be referred to the medics
for authorisation. 4. All clinical samples with a CT ≥40 should be reported as Not Detected
(ND) 5. Write the result (or use appropriate stickers) on the request forms as
SARS-CoV2 Nucleic Acid Detected/ Not Detected. CT values for positive samples are to also be recorded.
6. Samples testing positive for SARS-CoV2 with a CT value >35 must be repeated via the confirmatory assay, see Appendix 2.
7. Results are to be recorded On the RESPL test set within the Coronavirus result field. All other markers are to be left blank.
8. Report positive results as D6 (RNA Detected 2019 nCoV) with a report comment SARS-CoV2 RNA : Detected
9. Report negative results as ND with a report comment SARS-CoV2 RNA: Not Detected
10. The medics have requested that the Laboratory excel spreadsheet
(sent out daily) is populated prior to reporting results onto LIMS to ensure results are reported promptly to health protection.
11. Detected results are highlighted as RED and results that are being confirmed are highlighted in YELLOW
12. Once populated the list must be emailed to the designated staff member who has been tasks with the SARS-CoV2 admin duties.
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11.3 Inputting results into LIMS
1. In LIMS, open the WORKSHEET CONTROL module and enter the
code RESPLU to create a respiratory Luminex worksheet. Refer to CDVLSOP 007 and CDVLSOP 003 for worksheet generation and
result entry guidelines.
2. Manually select the episode numbers to add to the worksheet (or scan in using the request forms). Try to match the sample order on the
worksheet to the order in which the samples were tested. It is also useful to have the request forms in the same order
3. Open WORKSHEET RESULT ENTRY search for the RESPLU worksheet generated.
4. In the RESPT column enter OT as the platform used and batch fill. 5. Enter Detected (D) results as D6 within the CoV target field plus any
CT values. All other targets are to be left blank. 6. NOT DETECTED, RNAseP CT 35-37.9: enter the CT value into the
appropriate field on LIMS, suppress the field (highlight field, R-click and select suppress) and refer to the medics for authorisation this
must be done manually. 7. Enter Insufficient results (RNaseP ≥37.9) as INS within the CoV target
field plus the CT value within the RNaseP field. Results must be
referred to the medical queue for authorisation this must be done manually.
8. The Not Detected (ND) results can then be batch-filled for each column.
9. Comments for ND and D samples must be inputted within the comments box. Selected comment box (MRCOM) press F6 to expand
the comment box and copy and paste relevant comment SARS-CoV2 RNA: Detected
SARS-CoV2 RNA: Not Detected
10. Select ‘Multi-select Episodes’ Select All’ ‘Update’ ‘Authorise’.
11. Detected results will automatically go to the medical queue and Not detected results will go straight out.
12. Swab A is to be stored in the virology respiratory original storage
after sample preparation is complete
13. Swab b can be discarded after validation of a negative SARS-CoV2.
14. In the event that swab A is positive swab b (stored In the designated
secondary swab rack) is to be referred along with the extract to
genomics once confirmation testing, if applicable, has been
performed.
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12. AUTHORISATION
All detectable SARS-CoV2 results should be referred to the medics for
authorisation. All negative results are authorised by a BMS.
13. REPORT ISSUE (Interim/Final/Additional)
All results are reported on LIMS using the coronavirus results box on the
RESPL panel. All positive results with a CT >35 are repeated the same day through the screening assay and second line confirmation test - the screen
result issued as interim to allow appropriate action.
14. REPORTING TO OTHER DEPARTMENTS
All SARS-CoV2 positive results are referred to Genomics for sequencing.
15. REFERRAL TO REFERENCE UNITS
Refer to Appendix 1 for further information.
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Add 5l of extract to 20l Master Mix for each test
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Target Date Performed Operator
SARS- CoV2 +RNP ABI Fast
Volume per
10 20 30 40 50 60 70 80 90 100
Virus Fast Mix 62.5l 125l 187.5l 250 312.5 375 437.5 500 562.5 600
Batch
Primer Probe mix Batch
7l 14l 21l 28 35 42 49 56 63 70
Water 130.5l 261l 391.5l 522 652.5 783 913.5 1044 1174.5 1330
batch
Total volume 200l 400l 600l 800 1000 1200 1400 1600 1800 2000
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Add 5l of extract to 20l Master Mix for each test.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Target Date Performed Operator
SARS-CoV2 Confirmation ABI Fast
Volume per
10 (Sarbecco)
10 (Conf)
20 (Conf)
Virus Fast Mix 62.5l 62.5l 125l
Primer Probe mix 7l 5.5l 11l
Water 130.5l 132l 264l
Total volume 200l 200l 400l
Reagent Batch Number
Expiry Date
Primer Probe Mix Sarbecco)
Primer Probe Mix (Conf)
ABI Fast Virus Mix
Water
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16. Appendix 1- Additional Guidance
Essential Reading
CDVSOP 201 Detection of SARS-CoV2 using the ABI 7500 Fast
Morning Tasks
Re-stock lysis buffer
Re-stock small lysis
Dispose of negative swabs from previous day
Ensure all positive SARS-CoV2 samples (extracts + swab in lysis)
from the previous day have been located and stored in the
designated rack within the idolisation fridge for sequencing.
Waste Management
Store small lysis and extracts
Expected Sample: - Currently we are only expecting to receive 2 throat swabs
Receiving samples
Samples will be sent to the laboratory via courier or via designated
transport from within UHW
Couriers will deliver samples to a designated area within the
Virology Specimen Reception
When receiving samples from the community a yellow bin/ orange
cardboard bin containing waste will also be delivery by the courier.
Please see community waste for further information on disposal
Samples from A&E (If required)
If demand increases regular collection from A&E may be required
Take a sample transport box with you, all samples are to be
transported back to the laboratory within the transport box.
Samples for biochemistry testing may also have been taken it’s the
BMS responsibility to transport these to biochemistry. It is expected
that the clinicians have phoned ahead but this may not always be
the case. Bring the samples back to the lab if staff refused to
accept them.
All staff member should have access to A&E via their identification
badge
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Sample processing
Samples are to be processed in the Virus Isolation Laboratory
COVID-19 samples are transported under UN3373 Biological
Substance Cat B (Diagnostic Specimen) packaging legislation and
regulations. Samples should be packaged in a way that allows any sample package
to be opened safely in a laboratory area as per any other delivery. Even if the sample is suspected of containing CL3 pathogens the sample is
packaged in a way that prevents exposure to the micro-organisms upon initial receipt and inspection of the contents. The entire unopened package
should only enter CL3 if damaged and leakage of a sample is suspected at the point of receipt into the laboratory
For those laboratories that have a class 1 cabinet within the laboratory (if volume of samples allows) they could use this for opening the packaging
and follow steps outlined below using the cabinet as an additional control. This would need to be covered by local risk assessment. Risk assessment
should include contamination procedure for the cabinet if needed.
Processing samples in Class 1 Cabinet 1. Receive samples into designated area within specimen reception
2. Transport samples to Virus Isolation to begin processing in class 1 safety cabinet
3. Unpack samples out of cardboard box on the bench as per normal sample practice
4. Within the class 1 cabinet unpack sample from plastic container, It should still have a minimum of 2 barriers to the potentially
infectious material. i.e. the sample bag and primary container. 5. Match sample demographics with the request form. 6. Number 1x dry throat swab for SARS-CoV2 testing. Swabs are to
be broken into a corresponding numbered small lysis buffer and
incubated for a minimum of 10 minutes.
7. Snapped swabs are to be stored in a designated SARS-Cov2 sweetie
jar, all unsnapped swabs are to be stored in a separate labelled
sweetie jar.
8. Swabs can be discarded after validation of a negative SARS-CoV2.
9. Request forms are to be directed to a designated staff member for
data input and population of the laboratory list.
10. Discard boxes as normal process. (Hays DX to be returned in red
sacs for cleansing and reuse)
11. All waste generated must be disposed of in orange bags. After
validation of a negative SARS-CoV2 result sweetie jars of snapped
swabs can be disposed of as normal.
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Booking on + Scanning
Request forms are to be booked on by a designated member of staff
IA- CV PAS
Requestor-MIC
Location- UHPHL-
Request are inputted with the REPL test set and assigned outbreak
number 98 on data input
An additional RESPL test set is needed for GP and ITU surveillance
requests
Place forms on designated clip i.e. am/pm
Scan request form using the generic scanning log in. Password
available on pc
Luminex
GP and ITU surveillance samples require additional respiratory screening.
SARS-CoV2 extract should be referred for testing the following morning.
Please see comments below to be added when reporting results.
Biofire The medics may require rapid respiratory screening on some patient
samples. This will be discussed on a case by case basis
Transport small lysis (throat swab only) in a sample rack to room
to conduct Biofire testing
Conduct Biofire testing as normal.
Report results. Store lysis buffer in the respiratory storage
Comments
Comment to be added to all Luminex/ Biofire results
This test does not include SARS-CoV-2 (novel coronavirus)
Comment to be added to routine CoV positives
Consistent with a normal mild seasonal (common cold) coronavirus
Community Waste
Please do not take waste labelled as Cat B INTO CAT 3.
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Waste from the community has been assigned as Cat B. When
delivered to the laboratory seal the lid (if not already sealed) sign
the relevant section on the box label.
Dispose of bin/ box in the yellow bins for orange waste bags. If
you’re unsure store the waste bin in specimen reception and label
SARS-CoV2 waste to be dealt with the following working day.
Public Health Wales Microbiology Cardiff Virology Status: Controlled
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17. Appendix 2 Guidance for SARS-CoV2
Confirmatory Testing
1. In house confirmatory testing is to be performed on patient samples
with a CT >35.
2. Locate primary throat swab (swab a) from the original respiratory storage / isolation fridge
3. Locate the secondary swab (swab b) from the secondary swab storage rack
4. Print 4 episode numbers for each sample that requires confirmation 5. Label 2 large lysis and 2 skirted tubes with the patient name and
corresponding episode number. Provide a clear orientation mark to distinguish between the primary (Swab a) and secondary (swab b)
swabs. 6. Star the lid of the extract tube to make it known there is no MS2 in
this extract. 7. Both swabs are to be extracted at 110ul with no MS2.
8. When confirming the result samples must be repeated using the Sarbecco mix alongside the confirmatory mix as below
Sarbecco mix (SARS-CoV2 E gene) Patient sample in duplicate (primary and secondary extract)
Negative extraction control in duplicate 10-3 control in duplicate
10-4 control in duplicate Water
Confirmatory mix (SARS-CoV2 RdRp+ SARS-COV1 RdRp)
Patient extract in duplicate (primary and secondary extract if requested) Negative extraction control in duplicate
SARS control in duplicate Water
9. SARS-CoV2 Confirmation mix can be located next to the Sarbecco mix in the pp storage container
10. SARS positive control is located in the freezer in molecular (next to the
cabinets) 11. Please see Confirmation worksheet for master mix preparation
instructions. 12. All reagents required should be removed from the freezer and allowed
to defrost completely before use. Do not mix batches of reagent. 13. Record all reagent batch numbers and expiry dates on the worksheets
as required. 14. All reagents should be mixed and centrifuged before use. The ABI Virus
Fast Mix is very viscous and needs to be well mixed by inverting several times before centrifuging.
15. Add ABI Fast Mix to the primer probe aliquots as per quantities on the
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worksheet.
16. Add molecular grade water to the mix as per quantities on the worksheet. Multiple aliquots should be combined.
17. Gently mix and pulse centrifuge constructed mastermix. 18. Mastermixes should be used within 15 minutes of preparation and not
left exposed to the light, due to possible degradation of the fluorescent
probes 19. Set up PCR plate as normal and transfer to the amplification room
Note: Remove laboratory coat and wash hands before leaving the
laboratory.
20. Select the SARS-CoV2 testing template on the ABI, the template is available on ABI 1-3
21. Program episode number in full 22. Assign SARS-CoV2 E gene + RNaseP to repeat screening tests
23. Assign SARS-CoV2 RdRp+ SARS-COV1 RdRp to confirmation tests 24. Start Run- run approximately 45 minutes
25. Analyse data as normal 26. Check the thresholds are correct, edit threshold if required.
Thresholds should be set as :
SARS-CoV2 E gene (0.2) RNaseP (0.1)
SARS-CoV2 RdRp (0.2) SARS-COV1 RdRp (0.2)
27. Check each well individually by target. Note any samples that cross
the threshold (CT ≤40) for SARS-CoV2 Egene, SARS-CoV2 RdRp, SARS-COV1 RdRp .
28. Positive results for SARS-CoV1 are not expected, all positive results are to be discussed with the consultant virologist
29. All clinical samples should have an RNaseP signal. Make a note of samples that have no RNaseP signal, RNaseP CT 35- 37.9, or RNaseP
CT>37.9. 30. If any plots are not clear inform a Senior BMS.
31. Insert a pen drive into the USB port on the side of the laptop.
32. To export results select Print Report from the main menu at the top of the results page.
33. Transfer result report to the 2019 nCoV results folder. G drive: Virology: Virology Molecular Testing: 2019 nCoV
34. Input SARS-CoV2 E gene positive control and confirmation control CT value into the Medlab software, any result flagged in the red area
must be brought to the attention of a senior member of staff. 35. Expected CT range for Confirmation positive control should fall
between 33-35 36. Result interpretation and reporting see the table below
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Target
Report Report Comment Medic SARS-CoV2 E
gene SARS-Cov2
RdRp SARS -CoV1
RdRp
Result Combination
Positive Positive Negative D SARS-CoV2 RNA:
Detected
Refer result to the medical
queue
Positive Negative Negative D SARS-CoV2 RNA:
Detected
Inform Medic Confirmation assay is Not
detected
Negative Negative Negative ND SARS-CoV2 RNA: Not
Detected
Discuss with the medic as
screening assay did not
repeat
Pos/Neg Pos/Neg Positive Discuss with
the medic
NOTE: Ct values above 35 within the SARS-CoV2 Egene assay (although
likely to be true positives) because we are below the 100% level of detection of the assay, it is likely that in a proportion of samples the rdRp will NOT
confirm due to a reduced level of sensitivity (approximately 10 fold). In those instances it is likely that only the E gene screening assay will be
positive. Interpretation of these low level results will depend on clinical history and history of contact. Please refer results to the medics for
interpretation.
37. Swab A is to be stored in the virology respiratory original storage
after sample preparation is complete
38. Swab b can be discarded after validation of a negative SARS-CoV2.
39. Swab b is to be refered along with the extract to genomics for
sequencing.
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18. Appendix 3 SARS-CoV2 Testing Record
Date Episode Number Luminex
Result
SARS-CoV2
Result
Tested
bY