Detection of 2 SARS-CoV2 using the ABI 7500 Fast

Public Health Wales Microbiology Cardiff Virology Status: Controlled

Date Authorised: 13 March 2020 Document and Version: CDVSOP 201.2 Page 1 of 27 If printed, this document is only valid for today 10 April 2020 unless authorised as a controlled copy

-

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.



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.



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



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.



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.



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.



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



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.



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.



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



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.



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.



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).



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)



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



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.



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.




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.



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.



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



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



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



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.



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.



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.



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



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



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.




39. Swab b is to be refered along with the extract to genomics for

sequencing.



18. Appendix 3 SARS-CoV2 Testing Record

Date Episode Number Luminex

Result

SARS-CoV2

Result

Tested

bY

Documents

Detection of 2 SARS-CoV2 using the ABI 7500 Fast