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1
Purpose
To define the process for implementing SARS-Cov 2 RT-PCR testing using nasopharyngeal swab testing. The
tests will be from individuals for fit-to-fly and day 5&8 testing.
2
Scope
This procedure applies to all work carried out by Lamellar Ltd employees, including temporary / contract
workers who are performing operations/tasks for the benefit of Lamellar Ltd. This protocol will cover all
aspects of the testing procedure from collection of swab tests to analysis of results.
3
Responsibility
All members of staff are responsible to ensure results are reported correctly and submitted on time. Staff
are also responsible for receipt of tests and making sure none are misplaced during any stage of the process.
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Initial Preparation of Samples
1. NPS samples should be collected every morning from the designated Lamellar Collection Point which can
be found in the carpark next to the hazardous waste disposal.
2. Once collected the samples can be taken to lab A.
3. The outer package can be removed, and the box can be discarded into ordinary bins.
4. The inner sample bag can then be removed and placed into hazardous waste.
5. Samples can then be racked in the coloured 96-tube racks found near the oven in lab A.
6. Once samples are in the rack, they can be put into the oven in lab A for 30-45 minutes.
7. The samples are then ready to be vortexed for ~30 seconds.
8. Samples can then be moved into lab B for testing.
SOP
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5
Testing Procedure
The VirPath Xtract Viral DNA/RNA Extraction Kit is intended to be used for rapid total nucleic acid extraction from
nasopharyngeal swabs and other biological fluids. The kit has been specifically validated for the extraction of viral
RNA from specimens containing the SARS-CoV-2 virus.
Kit contents
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VirPath Lysis Buffer
VirPath Target Pure Beads
Required equipment
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1 x 2 to 125ul ClipTip pipette
1 x 15 to 1250ul ClipTip pipette
1 x 300ul multichannel
1 x 1000ul pipette
1 x 200ul pipette
1 x20ul pipette
PCR-clean filtered tips
96-well deep well plates
Magnetic separation rack capable of accommodating 96-well deep well plates
Consumables
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100% isopropanol, molecular biology grade
80% ethanol, molecular biology grade
Buffer EB or equivalent buffer saline solution (10 mM Tris-HCl, ph8.0)
Water, nuclease free molecular biology grade
Storage and Handling
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Upon receipt, store VirPath Target Pure beads refrigerated at 4°C (do not freeze). VirPath Lysis Buffer is
stable at room temperature for 12 months.
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Extraction Procedure
Before beginning, remove the VirPath Target Pure beads from storage and place at room temperature for 30
minutes. Prepare a fresh 80% ethanol solution (800 ul per sample are required).
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2.
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5.
6.
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17.
Aliquot 200 μl of sample into a fresh 1.5 ml tube or well of a 96 deep well plate. (1250ul pipette)
Add 100 μl of VirPath Lysis Buffer to each sample. (Multichannel pipette)
Vortex the tube(s) / plate for 10 seconds and incubate at room temperature for 10 minutes.
Add 40 μl of thoroughly vortexed room-temperature equilibrated VirPath Target Pure beads to each
sample. (Multichannel pipette)
Add 270 μl of 100% molecular grade isopropanol to each sample and mix gently by pipetting up and
down 10 times, taking care to avoid the formation of bubbles. (Multichannel pipette)
Place the tubes / 96 deep well plate on the magnetic stand for 10 minutes at room temperature to pellet
the beads on the side of the tubes/wells.
Keeping the tubes / 96 deep well plate on the magnetic stand, slowly remove and discard the
supernatant, taking care not to disturb the pelleted beads.
Add 400 μl of 100% molecular grade isopropanol to each tube/well and incubate at room temperature
for 30 seconds. (Multichannel pipette)
Keeping the tubes / 96 deep well plate on the magnetic stand, slowly remove and discard the
supernatant, taking care not to disturb the pelleted beads.
Add 400 μl of 80% ethanol to each tube/well and incubate at room temperature for 30 seconds.
Repeat steps 9-11 for a total of two 80% ethanol washes. (Multichannel pipette)
Keeping the tubes / 96 deep well plate on the magnetic stand, slowly remove and discard the
supernatant, taking care not to disturb the pelleted beads. (Multichannel pipette)
Use a 10 μl multichannel or single channel pipette to remove any residual liquid from the tubes/wells.
Keeping the tubes / 96 deep well plate on the magnetic stand, incubate at room temperature with open
lids for 3-5 minutes or until the beads are dry.
NOTE: it is important to avoid over-drying of beads, as this can result in a significant loss of RNA/DNA
recovered.
Remove the tubes / 96 deep well plate from the magnetic stand and resuspend the dried beads in 52 μl
of Buffer EB or equivalent buffer saline solution (10 mM Tris-HCl, ph8.0) by pipetting up and down 10-15
times, taking care to avoid the formation of bubbles. (Multichannel pipette)
Incubate the tubes / 96 deep well plate for 2 minutes at room temperature.
Place the tubes / 96 well plate on the magnetic stand for 3-5 minutes at room temperature to pellet the
beads on the side of the tubes/wells.
NOTE: if after 5 minutes the beads have not entirely pelleted to the side of the tube, add 10 μl of Buffer
EB or equivalent and wait 3 more minutes for beads to pellet. Keep incrementing the amount of Buffer EB
or equivalent added if beads are still not pelleting to the side of the tube until the solution is entirely
clear of beads.
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7
RT-PCR
Kit contents
VirPath qRT-PCR Master Mix
Multiplex primer / probe mix
RPP30 Negative Control
2019-nCoV nucleocapsid and envelope genes Positive Control
ROX Reference Dye (10x)
Upon receipt, store all reagents at -20°C.
Equipment
1 x 125ul ClipTip pipette
1 x 20ul pipette
PCR-clean filtered tips
1.5 / 2 ml cold block (or access to ice)
96 well cold block (or access to ice)
qPCR Instrument (4 colour) (Quant5studio)
96 well plate and optical seal compatible with qPCR instrument
96 well plate compatible vortexer
Consumables
Molecular grade RNAse free water
SOP
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8
SOP
Revision:
Page: 8 of 8
Approved Date:
Effective Date: 16/06/2021
Approved by:
Doc Holders List:
Training
Statement of Use: Verify status before each use