Policy # MI MD COVID Page 1 of 40 Department of ......Department of Microbiology Quality Manual...

Department of Microbiology

Quality Manual

Policy # MI_MD_COVID

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Version: 5.4 CURRENT

Section: Molecular Diagnostics Procedures Subject Title: SARS-CoV-2 PCR by AllplexTM Seegene

Prepared by QA Committee

Issued by: Laboratory Manager Revision Date: 4/26/2021

Approved by Laboratory Director:

Microbiologist-in-Chief

Next Review Date:

Uncontrolled When Printed

UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY

NOTE: This document is Uncontrolled When Printed. Any documents appearing in paper form that do not state "CONTROLLED COPY " in red print are not controlled and should be checked against the

document (titled as above) on the server prior to use. Management System\UHN_Mount Sinai Hospital Microbiology\Standard Operating Procedures\Molecular Diagnostics Procedures\

TABLE OF CONTENTS

Introduction ..................................................................................................................................... 3

Collection and Transport & Storage ................................................................................................ 3

Procedure ......................................................................................................................................... 6

General Precautions: ........................................................................................................................ 6

Pre Board Preparation: ..................................................................................................................... 7

Microlab STARlet ............................................................................................................................ 8

CFX96TM

Amplification and Detection .......................................................................................... 20

PCR Analysis ................................................................................................................................. 21

Interpretation ................................................................................................................................. 28

Seegene Report Interpretation ........................................................................................................ 28

General Seegene Reporting Process ............................................................................................... 29

Seegene Reporting Process for Repeat Samples ............................................................................ 31

Reporting ....................................................................................................................................... 32

PRELIMINARY RESULTS (for UHN/MSH/STAT patients only): ....................................... 32

FINAL RESULTS ........................................................................................................................ 33

Cleaning ......................................................................................................................................... 36

Quality Control .............................................................................................................................. 37


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Section: Molecular Diagnostics Procedures Subject Title: SARS-CoV-2 PCR by AllplexTM

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NOTE: This document is Uncontrolled When Printed. Any documents appearing in paper form that do not state "CONTROLLED COPY” in red print are not controlled and should be checked against the


Troubleshooting ............................................................................................................................. 38

Related Documents ........................................................................................................................ 38

References: .................................................................................................................................... 38

Record of Edited Revisions ........................................................................................................... 39


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Introduction

The 2019–20 coronavirus outbreak is an ongoing public health emergency of international concern

involving coronavirus disease 2019. It is caused by SARS-CoV-2, first identified in Wuhan, Hubei,

China.

SARS-CoV-2 is closely related to the original SARS-CoV. It is thought to have a zoonotic origin.

Genetic analysis has revealed that the coronavirus genetically clusters with the genus

Betacoronavirus, in lineage B of the subgenus Sarbecovirus together with two bat-derived strains. It

is 96% identical at the whole genome level to other bat coronavirus samples (BatCov RaTG13). In

February 2020, Chinese researchers found that there is only one amino acid difference in certain

genome sequences between the viruses found in pangolins and those from human patients, implying

that pangolins may have been an intermediate host.

Allplex™ 2019-nCoV Assay (100T) is a qualitative in vitro test for multiple detection of 3 types of

gene (RdRp gene, Envelope gene, and Nucleocapsid gene).

Collection and Transport & Storage

Note: Nasopharyngeal (NP) swabs, throat swabs, saliva, sputum, Bronchial Alveolar Lavages

(BALs), EDTA plasma/serum, stool and breast milk are verified sample types for this assay.

I. Respiratory samples

Respiratory samples such as Sputum, Throat swabs, Nasopharyngeal (NP) swabs

and Bronchial Alveolar Lavages (BAL) are tested.

Sputum and BAL can be stored at 4°C for up to 72 hours after collection in sterile

container.

NP and Throat swabs can be stored 4°C for up to 72 hours after collection in

Universal Transport Media (UTM™) or equivalent.

If the specimen is not going to be tested within 72 hours of collection, then it

should be stored at -70°C or lower.

II. Blood samples

Specimen collected in serum separator tube/red top tube or EDTA tubes may be

tested

Follow the tube manufacture’s processing instructions for serum and plasma

collection tubes. Gravity separation is not sufficient for specimen preparation.

https://en.wikipedia.org/wiki/Public_Health_Emergency_of_International_Concern

https://en.wikipedia.org/wiki/Coronavirus_disease_2019

https://en.wikipedia.org/wiki/Severe_acute_respiratory_syndrome_coronavirus_2

https://en.wikipedia.org/wiki/Wuhan

https://en.wikipedia.org/wiki/Hubei

https://en.wikipedia.org/wiki/Severe_acute_respiratory_syndrome_coronavirus

https://en.wikipedia.org/wiki/Zoonosis

https://en.wikipedia.org/wiki/Betacoronavirus

https://en.wikipedia.org/wiki/Severe_acute_respiratory_syndrome-related_coronavirus

https://en.wikipedia.org/wiki/Genome

https://en.wikipedia.org/wiki/Pangolin

https://en.wikipedia.org/wiki/Intermediate_host


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Separated serum /EDTA plasma can be stored in 4°C for up to 72 hours after

collection. If the specimen is not going to be tested within 72 hours of collection,

then it should be stored at -70°C or lower.

III. Placenta/ conception swabs

Placenta/conception swabs can be stored at 4°C for up to 72 hours after collection



IV. Stool samples

Stool can be stored at 4°C for up to 72 hours after collection in sterile container.



V. Saliva samples

To maintain optimum viability, the specimen should be stored and transported at

2-8°C. If transport to the laboratory will be delayed, specimen should be frozen at

-70°C or below and shipped on dry ice.

Acceptable for testing up to 48hrs at room temperature

VI. Other specimen types (e.g. urine, tissue, miscellaneous samples, etc.) need to be

approved by the Microbiologists

Materials, Equipment and Facilities:

Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG)

Specimen Preparation area: Biosafety Cabinet –2.5 level

Microlab STARlet

CFX96TM

IVD

Nimbus-96 Deep Well Micro Plate

1.5ml micro tube

Optical Flat 8-Cap strips

Hard-Shell 96 Plate, White shell, White wells

High volume Tips (1000 µL)

Standard Volume Tips (300 µL)

Sample and reagent racks 1.5 ml RNase-free microcentrifuge tubes

Variable volume Rainin pipettes: 1 to 20 µL, 10 to 200 µL, 100 to 1000 µL


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Reagents:

AllplexTM

2019-nCoV Assay

STARMag 96×4 Cartridge Kit

i. STARMag 96×4 Universal Cartridge Kit

ii. STARMag 96×4 C-Type Cartridge Kit

iii. STARMag 96×4 L-Type Cartridge Kit

< µL > Original version C version L Version A

PK 10 Not required Not required Not required

IC 10 10 10

Lysis Buffer 150 150 350

Sample 200 200 200

Beads 10 10 Included in LB

Binding buffer 586 586 Included in LB

Washing Buffer 1 490 490 350

Washing Buffer 2 490 490 350

Washing Buffer 3 500 500 Not required

Elution Buffer 100 100 100

External controls: Positive external controls (positive for all E/N/R genes) and Negative

external controls (negative for all E/N/R genes) to be run for each new lot/shipment if QC

fails


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Procedure

General Precautions:

There must be separate PCR work areas:

Clean room

Specimen preparation room-2.5 level lab

Powder-free Gloves should only be in use in PCR areas.

Change gloves frequently and keep tubes closed whenever possible.

Prepare Working 1% sodium hypochloride daily.

Specimen Preparation Supplies and equipment must be dedicated to Specimen Prep Area and

not used for other activities and never used in Clean Room.

Change lab coats and gloves between work areas.

Use only Aerosol Resistant Tips (ART)

Use only sterile RNase-free, DNAse-free microtubes

Thaw components completely at room temperature.

Clean PCR work areas (Clean Room and Specimen Preparation Area) bench tops and

equipment after each shift.

Do not pool reagents from different lots or from different tubes of the same lot.

Do not use the product after its expiry date.

Sample Preparation:

1. Sputum:

Viscous sputum sample should be treated with Sputolysin prior to testing at a 1:2

dilution.

2. Stool:

Pipette 500 µL PCR grade water or Universal Transport Media (UTM) into 1.5mL

tube

Take a swab from Cepheid VRE kit to swab the stool

Insert swab into the stool specimen and roll the swab over the specimen to be fully

coated.

Note: make sure that swab is brown but not including a large amount of fecal

material

Vortex the stool resuspension tube at the highest setting for 15 seconds. Ensure full

vortex is performed during mixing.

Centrifuge stool resuspension Tube at a minimum of 2,000 x g (4643 rpm) or 13,200

rcf for a minimum of 30 seconds.


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Pipette 300 µL of supernatant into another new 1.5mL tube for subsequent testing

3. Tissue

If PCR is performed from tissue in formalin, first take the tissue out of formalin and

place it in UTM before grinding.

If there is significant amount of formalin, dilute out further by placing in second

UTM vial before grinding.

a portion of the ground up sample gets placed into a freezer box inside MIRT16 fridge

the remaining ungrounded sample goes into MIRT17 fridge

Scan request form into donor folder

4. Saliva

Dilute 1:1 with saline (ex. 300 µL saliva + 300 µL saline)

Pre Board Preparation:

1. Make sure the specimen ID label is just underneath the rim, straight up and no fold made

2. Make sure there is at least 300 µL in the 1.5ml conical tube

3. Place samples into 65°C for 30 minutes

4. After 30 minutes, take the samples out of the incubator and bring samples to BSC

5. Load the tubes onto Seegene sample carriers and CAREFULLY uncap the tubes

Note: Please avoid contamination between tubes. Change gloves if needed

6. Make sure specimen IDs on the tubes are fully visible for automatic barcode scanner to read.

(If they are well attached on the tubes, all barcodes will be read.)

Note: Leave the sample carries in the BSC till ready to load onto Seegene


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Microlab STARlet

1. Click Maintenance button

2. Select proper maintenance (daily or Weekly) and click Start button

3. Following the steps on the screen to finish the necessary maintenance process.

4. Close the maintenance screen

5. Double click SLnCoV icon on the desk top

6. The following message window pops up, click OK button

Start button


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7. Click LAUNCHER RUN

8. Select One Step for 2019-nCoV

Note the following procedure changes when using the C or L Kit

C-Kit procedure L-Kit procedure A-Kit procedure

1. There will be no PK

2. Before use, you should aliquot

1. There will be no PK in version

L


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1.8ml magnetic beads from the

big bottle given into four empty

2ml tubes. Make sure to vortex

the magnetic beads bottle VERY

thoroughly WELL to remove any

aggregation before the dispense.

If a partial and new kit are

loaded, 2 tubes are required

If only a new kit is loaded, 1 tube

is required to be loaded.

3. Start a protocol

4. Scan the extraction cartridge

barcode as it (see a picture

below) prompts after starting the

protocol.

5. Remaining process is the same

2. Magnetic beads are in Lysis

buffer so make sure to mix

them well before the use by

pipetting up and down in the

liquid (NOT above the liquid

due to bubbles generated) by

1000 µL pipette or the lysis

buffer tube itself can be

separated. Before opening the

plastic cap, take the lysis buffer

tube ifself out and gently invert

it to mix them well.

3. Start a protocol

4. Scan the extraction cartridge

barcode as it(see a picture

below) prompts after starting

the protocol.

Remaining process is the same

9. Prepare Waste Bag-Make sure the tip waste is in position.

If not, place it correctly.

Click Next

10. Prepare 1000 µL Tip

Place the 1000 µL filter tips to the required position according to the screen display

There are 7 positions for the 1000 µL tips

Click Next


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11. Prepare 300 µL Tip

Place the 300 µL filter tips to the required position according to the screen display (4

positions)

There are 4 positions for the 300 µL tips

Click Next

12. Prepare Small Tube Rack Carrier

Place the small tube rack carrier at Tack No. 24

After confirming PCR information, place the PCR reagent in the small tube rack 1 or

1,2

Click Next


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13. Prepare IC

Place RP-VIC on appropriate position in the small tube rack 1

Click Next

14. Prepare PK

Prepare PK if required

Prepare the PK:

Add 2600 µL of Proteinase Buffer Universal PB in to Universal Proteinase K to

lyophilize the powder and let the bottle stand still for 10 minutes before use

Note: Instrument needs a little more than what the system calculates, please top it up

(i.e. if the system say 1170 µL, top it up to 1200 µL; if it says 270 µL, top it up to

300 µL)

Type the total sample number in the Sample Qty window, the necessary volume of

PK is calculated by the system.


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Pipette required volume of PK into the 1.5 ml sterile microtube according to the

number of the samples and place it to the designated position in the small tube rack

1

Click Next

Note: Prepare PK before running the instrument. The exact volume will be indicated

on the Work List

15. Prepare Magnetic Bead

Re-suspend the bead tube with hand tapping and then vortex for more than 1

minute

1st Bead position:

Place the Bead tube on the designated position in the small tube rack 1.

2nd

Bead position:

If the total number of prepared samples exceeds the remaining samples covered by

previous cartridge, add a new tube of magnetic beads (see the picture below)


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Click Next

Note: Must pair extraction cartridge and bead location. (#1 to #1, #2 to #2)

16. Prepare Extraction Reagent

Add absolute ethanol to the Wash Buffer cartridge before using for the first time.

Refer to the Tub Label of WB2.

Universal Kit & C-Type Kit: add 48mL of ethanol into WB2

L-Type Kit: add 38mL of ethanol into WB2

A Kit: add 15mL of ethanol into WB1 and 48mL of ethanol into WB2

Place the extraction reagent cartridge at Track No.18

After completing work list, scan the reagent barcode at the Reagent Barcode Page

and load the required extraction reagent to the designated position (see picture

below)

Click Next


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17. Prepare 96 DWP

Place the 96DWP on the designated position.

When using barcoded 96 DWP, the barcode of DWP should be placed to right side

of plate.

Click Next

18. Prepare PCR Plate

Place the Plate Bio-Rad White on the designated position.

After confirming PCR information, place the Plate Bio-Rad White in the plate 1

and 2.

When the samples exceed 96 well in plate, it automatically turns over to plate 2.

Note: Normally the sample number is less or equal to 96, only need to load PCR

plate 1

When using barcoded PCR plate, the barcode of plate should be placed to right side

of plate

Click Next


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19. Prepare Sample

Prepare each sample on tubes, referring to sample type and tube size

Then place them into appropriate position

Each tube must contain more than a guided volume of samples (Refer to Sample

preparation guide)

Insert the stop barcode tube into the following location after loading all the

samples.

Sample Preparation Guide

Minimum Volume (µL)

Tube Specimen

1.5mL 300

12 mm 800

16 mm 2000

20. Prepare Sample Carrier

Place the 32 sample carriers at Track No.13, 14 and 15

If Sample Unload is turned “ON”, after sample dispense, sample carriers are

automatically unloaded

Click Worklist


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Read specimen IDs:

I. Automatically read specimen IDs :

Click Barcode button on the top right hand side (no need to type

quantity of the samples)

II. Manually scan specimen IDs:

Manually scan specimen IDs by using hand barcode provided (need to

type the quantity of samples)

Note: In case you let the system read the sample IDs automatically, if ID labels are

not well placed, there is a high chance it will not be read. Therefore, have

worksheet with all IDs that you loaded on the system ready so any missed

specimen IDs you can manually scan IDs accordingly

21. Scan extraction reagent barcode. When there are not enough reagents left in the 1st cartridge,

there will be a pop-up window asking you to place the second set of reagent cartridge. Click

OK and then scan the barcode from the second reagent cartridge.

22. Click Next


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23. Scan PCR reagent barcode. When there are not enough reagents left in the 1st PCR reagents,

there will be a pop-up window asking you to place the 2nd

set of PCR reagents. Click OK and

then scan the barcode from the second one.

24. Click Next

25. Place each PCR reagent accordingly. Make sure to completely thaw, vortex (3~5 seconds) and

quick spin them down. If there are not enough PCR reagents left from the 1st kit, you need to

place new PCR reagents in the 2nd

row as you scanned the barcode.

26. Click Next

27. PCR plate information tells you how many 8 strip caps you will need at the end. Click Next


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28. Follow the following check list to make sure everything is ok

1000 uL Tip

300 uL Tip

Sample

Carrier

Extraction

Reagent


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29. Click Run

30. At the last pop up screen, be sure that you placed everything in the correct position and mark

the check box as you read it line by line once you followed.

31. Do not leave as you will need to indicate tip positions. In 2~3 minutes, there will be pop up

screen for you to indicate tips location and make them exactly identical to where you placed.

Click and drag onto to the gray-out 1000 µL tips spot to make sure it turns to brown

indicating that all 1000 µL tips are present, click OK

Click and drag onto to the gray-out 300 µL tips spot to make sure it turns to brown

indicating that all 300 µL tips are present, click OK 32. Once extraction is completed, pop-up window saying Method Completed, click OK button

Note: This will generate the plrn file to the Biorad Thermocycler

CFX96TM

Amplification and Detection

1. On the desktop chose plrn (starlet-PC)

2. Choose the run you want to start by double clicking on it Choose open lid and load plate

into thermocycler

3. Choose open lid and load plate into thermocycler

4. Choose close lid

5. Go to plate tab

6. Go to edit select and check the sample IDs and make sure it is correct run


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7. Click OK

8. Choose next

9. Start Run

10. Name the run and save to current folder

PCR Analysis

1. Check that the signal at the beginning of cycling is OK

2. Check that the curves are sigmoidal

3. In the top menu bar, Click Tools

4. Select Seegene export


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5. Select COVID-19 RAW DATA

6. Click OK

7. Click “X” at the top right corner of the window to close file

Click NO to the following Message Window “Do you want to save the changes…:,

Do not save changes, store raw data as is


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8. Click “X” at the top right corner of the window to close the Bio-Rad CFX program

9. On the desktop, double click 2019-nCOV Viewer

10. The following message window pops up, click “OK”

11. Open Raw Data

a. Click File->Open

b. Click Desktop

c. Select COVID-19 RAW DATA folder

d. Click Open


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Open current run by date and time

Open Nimbus-yes

Desktop-plrn(STARLET-PC)-open your run (need to scroll right)

Select all samples (well info)

12. Select Quantstep4, Click Open

13. Select current run by date and time and click Open


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14. Nimbus/STARlet Setting window pops up, click Open

15. Click Desktop, select plrn(STARLET-PC) – Shortcut, Click Open

16. Select your run (need to scroll right), click Open


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17. Create the results in PDF file

a. Select all samples by checking well info

b. Click Print Icon

c. Type the name of the operator

d. Click PDF

e. Click Desktop button

f. Select COVID PDF RESULTS

g. Click Open

b

d

c

a

e f

g


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h. Type the file name including the date and operator’s name

i. Click Save

j. Must wait for the complete message window pops up

k. Click OK

l. On the desktop, double click the folder of COVID PDF RESULTS

m. Open your PDF result and print

Auto-Transfer of Results

1. In the 2019-nCOV Viewer uncheck any sample numbers that have to be exported

manually i.e.; BGI repeats that are negative, invalid non-NP, non-throat and non-

sputum samples, offline repeats

2. Create a CSV file by using File > Export CSV

3. Save file under the shortcut to the Seegene (eg. 1_Seegene1 shortcut on the desktop)

using the same name as the PDF file

4. Close 2019 nCoV-Viewer

5. Open bioftp program on the desktop

6. Drag CSV file from the Seegene folder from the left window to the right window


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Interpretation

When a successful run is completed (positive and negative controls yield expected corresponding results)

use the table below for sample interpretation.

Seegene Report Interpretation

Negative Internal Control readings in HEX (IC) may occur in strongly positive FAM, Cal Red 610

and Quasar 670 channels results. High signal for E/N/RdRP gene in the sample often deplete the

amplification/detection materials (eg. nucleotides), leading to reduced or absent Internal Control

signals.

E-gene RdRP-gene N-gene IC Interpretation

- - - + COVID-19 Virus NOT Detected

+ + + +/- COVID-19 Virus Detected

- + + +/- COVID-19 Virus Detected

+ + - +/- COVID-19 Virus Detected

+ - + +/- COVID-19 Virus Detected

- + - +/- See “General Seegene Reporting Process”

+ - - +/- See “General Seegene Reporting Process”

- - + +/- See “General Seegene Reporting Process”

- - - - INVALID

See “General Seegene Reporting

Process”Repeat in Neat for all non-NP,

non-throat, non-sputum samples


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General Seegene Reporting Process


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Seegene Reporting Process for Repeat Samples


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Reporting

Follow “General Seegene Reporting Process”.

PRELIMINARY RESULTS (for UHN/MSH/STAT patients only):

PRELIMINARY POSTIVE RESULTS:

Result in the isolate window as COVID-19 virus

In the Isolate comment, choose \DPCR & \ind

Status as PRELIMINARY

Inform all positive results according to COVID RESULT COMMUNICATION

Example:

COVID-19 virus

DETECTED by real-time PCR.

* * * * * * * * * * * * * * * * * *

(Low level detection)

Sample is being retested. Further report to follow.

Testing performed using the Seegene Allplex 2019-nCoV Assay.

NOTE: The Seegene Allplex 2019-nCoV Assay has been approved

by Health Canada for Emergency Use Access (EUA) and has

been verified by the University Health Network/Sinai

Health Microbiology Laboratory.


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FINAL RESULTS

NEGATIVE RESULTS:

Result in the test window using COVID-19 Virus Not Detected test comment

Choose from the keypad: }-NCS (Seegene)

Status as FINAL

When Seegene .csv file for a run is exported to LIS, all negative samples should auto-

finalize.

Example:

COVID-19 virus NOT detected by real-time PCR.

* * * * * * * * * * * * * * * * * * *





Health Microbiology Laboratory.\

POSITIVE RESULTS:

Result in the isolate window as COVID-19 virus

In the Isolate comment, choose \CVS+ (Seegene)

Status as FINAL

Inform all positive results according to COVID RESULT COMMUNICATION

Example:

COVID-19 virus

DETECTED by real-time PCR

* * * * * * * * * * * * * * * * * *





Health Microbiology Laboratory


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INVALID RESULTS:

Repeat in Neat for all non-NP, non-throat and non-sputum samples

Result in the test window using COVID-19 virus PCR test unable to be completed test

comment

Choose from the keypad: }INCS (Seegene)

Status as FINAL

Inform all invalid results to the ward /clinic

Example:

COVID-19 virus PCR test unable to be completed.

* * * * * * * * * * * * * * * * * * *

Test result is suggestive of inhibition of the Seegene

Allplex 2019-nCoV Assay PCR reaction. Please submit

another sample for testing if clinically indicated.




Health Microbiology Laboratory.


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FINAL RESULT for REPEAT of LOW LEVEL DETECTION:

Follow Seegene Reporting Process for Repeat Samples

CONFIRMED POSITIVE RESUTLS:

Follow FINAL RESULTS for POSITIVE reporting procedure.

INDETERMINATE RESUTLS:

Suppress COVID-19 virus isolate

Use the following Indeterminate test comment

Status as FINAL

Inform all updated confirmed results according to COVID RESULT

COMMUNICATION

Example:

Indeterminate for COVID-19 virus.

* * * * * * * * * * * * * * * * * * *

Results should be interpreted within the context of the

clinical signs, symptoms, and history of the patient.

INDETERMINATE result may indicate the presence of low levels

of virus, non-specific reactivity of the assay, or other unrecognized factors. Please submit a

follow-up sample if clinically indicated.

Testing performed using the BGI Real-Time Fluorescent

RT-PCR 2019-nCoV Assay.

NOTE: The BGI Real-Time Fluorescent RT-PCR 2019-nCoV Assay

has been approved by Health Canada for Emergency Use Access

(EUA) and has been verified by the University Health

Network/Sinai Health Microbiology Laboratory.

* * * * * * * * * * * * * * * * * *

DISCLAIMER:

NP swab, throat swab, sputum, BAL, saliva, EDTA plasma/serum, stool and breast milk

are verified samples for testing on Seegene.

Run all “non-verified” samples (tissue, placenta, CSF and other body fluids) as a batch at

the beginning or end of a run so that it’s easy to prevent these from being transmitted to

LIS.


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Follow General Seegene Reporting Process

Add the following disclaimer for non-verified sample types.

Its performance has NOT been validated on this specimen type.

Please take this into consideration when interpreting this result.

Cleaning

Routine Cleaning:

After each run:

After each Seegene run, wipe down with RNase Away and alcohol the Starlet platforms,

arms/probes that pick up the pipette tips to reduce potential cross-contamination.

Weekly:

Soak and disinfect sample racks in 10% bleach solution for 10 minutes and rinse thoroughly with

water. Let fully dry.

At shift end, perform cleaning protocol as outlined below:

Clean Room: Wipe down with RNAse Away or NucleoClean on paper towel, followed by distilled

water, and then 70% alcohol

Biological Safety Cabinet

Pipettes

Bench tops

Specimen Preparation Area: Wipe down with working 1% hypochloride (made daily), followed

by distilled water, and then 70% alcohol

Biological Safety Cabinet (BSC), pipettes, centrifuge, and bench top.

Seal and discard BSC waste.

Soak and wash sample loading racks weekly.

Amplification Area: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe,

followed by UltraPure water, and then 70% alcohol Wipe.

Seal & discard reaction microtubes into biohazard waste after each run.

Perform the cleaning procedure according the daily maintenance sheet.


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Quality Control

Reagent QCs:

An External Control (external to Seegene reagent kits) is used to monitor the isolation,

amplification and detection procedures. The result must correspond to expected value supplied by

the manufacturer.

External control should run with each new shipment or with each new lot.

Daily QCs:

Every run:

Each patient specimen must have an Internal Control (IC) added to monitor both extraction

and PCR inhibition.

A Positive Control is included and shows amplified signal in each corresponding channel

A Negative Control is included and shows negative signal in each corresponding channel

Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.

a. Do not release results pending resolution of QC failure.

b. Inform charge/senior technologist.

c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where

appropriate.

d. If the QC failure was due to a simple matter of position reversal or misplacement, the run

can be released (positive QC material yielded positive result, negative yielded negative

result) with approval from senior, charge or microbiologist.

e. If positive QC material yielded negative result, repeat the entire run.

f. If negative QC material yielded positive result, it may be due to cross-contamination from

adjacent positive sample within the run or carry-over contamination from previous runs via

equipment or the environment. Review procedure and equipment to establish and eliminate

potential sources of contamination.

g. The extent and nature of contamination can also be evaluated by comparing the positive rate

of the run with its expected positive rate.

h. If the contamination is extensive, it is necessary to quarantine/discard potentially

contaminated reagents and consumables and disinfect equipment and environment before

repeating the run.

i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being

positive or patient samples have higher than expected positive rate and these samples are


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often non-repeatable positives), it is necessary to have a thorough environmental

disinfection followed by swabbing to monitor.

j. Successful ending to a carry-over contamination may be indicated by QC results and patient

positivity rate falling back to the expected normal range and three negative environmental

swabs.

Troubleshooting

Refer to

For technical support Email description of errors and include files below as attachments to Seegene technician support staff as per Equipment Maintenance Procedure for the Starlet.

1. Trace logs (always) 2. Screenshot or exact error or error message (if applicable) 3. Abort logs (if applicable) 4. .pcrd file (result-based rather than mechanical issue)

Related Documents

Virology Accessioning Manual MI_MD_ACC

Training Checklist

References:

Seegene Sars-CoV-2 Allplex Assay package insert.

https://eportal.mountsinai.ca/MSHPresentations/public/paradigm/D0024510.pdf


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Record of Edited Revisions

Manual Section Name: SARS-CoV-2 PCR by AllplexTM Seegene

Page Number / Item Date of Revision Signature of

Approval

Update pre-lysis procedure to heat fix procedure

Remove email communication to microbiologists,

director, manager

Updated reporting section

March 16, 2020 Dr. T. Mazzulli

Add manually scan sample IDs

Add extraction reagent barcode process

Add PCR reagent barcode process

Update reporting and send-out section


Updated reporting to remove any indeterminate or PHOL

send out reporting.


Updated Collection and Transport & Storage:

Changed ‘4°C for up to 7 days’ to ‘4°C for up to 72

hours’ due to initial typo


Updated acceptable sample types

Added invalid reporting

April 14, 2020 Dr. T. Mazzulli

Updated acceptable sample types:

Added Placenta/ conception swabs Stool samples

Added the sample preparation for stool and viscous

sputum

Updated the reporting:

No need to report the individual gene


Updated Report:

No need to repeat samples with initial invalid results;

report final results of “INVALID”


Updated phrases for invalid reporting

Added back “No gene targets were detecte…..

Please submit another sample for testing if

clinically indicated.”

Updated to repeat all invalid non-NP/non-Throat/non-

sputum samples in neat

Clarified NP/Throat/Sputum/BAL/EDTA plasma and

serum are verified sample types for this assay.



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Page Number / Item Date of Revision Signature of

Approval

Added the disclaimer for reporting non-verified samples

Addition of C and L kit procedure.

Addition of new interpretation and report testing

algorithm for N-Gene only testing.

May 20, 2020 Dr. T. Mazzulli

Addition of Seegene troubleshooting guide link August 20, 2020 Dr. T. Mazzulli

Addition of Low positive algorithm for single gene

positives. Addition of low positive confirmed, or

indeterminate repeats from BGI. Addition of auto-

resulting instructions

August 24, 2020 Dr. T. Mazzulli

Full document review included in all updates. Bi-annual review conducted when no revision had

been made within 2 years.

Page Number / Item Date of Revision Edited by:

Addition of General Seegene Reporting Process,

Seegene Reporting Process for Repeat Samples;

Updated Seegene report interpretation table, reporting

rules and preliminary and final report statements in

test window and isolate window

Updated disclaimer for non-verified samples

Addition of routine cleaning after each Seegene run

December 31, 2020 Dorna Zareianjahromi

Updated Saliva testing info March 22, 2021 Dorna Zareianjahromi

Updated Tissue processing info April 9, 2021 Dorna Zareianjahromi

Updated the list of verified sample types with stool and

break milk included

April 9, 2021 Dorna Zareianjahromi

Minor formatting change April 11, 2021 Jessica Bourke

Update on a typo in the reporting part April 20, 2021 Oliver Li

Updated files required for troubleshooting with technician

support

April 24th

, 2021 Jessica Bourke

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