Policy # MI MD ADEBR Page Department of Microbiology ... · Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added together

Department of Microbiology

Quality Manual

Policy # MI_MD_ADEBR

Page

Version: 1.0 CURRENT 1 of 24

Section: Molecular Diagnostics Procedures Subject Title: Adenovirus Qualitative PCR Bio-Rad

CFX96 Prepared by QA Committee

Issued by: Laboratory Manager Revision Date: 4/12/2021

Approved by Laboratory Director:

Microbiologist-in-Chief

Annual Review Date: 4/12/2023

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 are not stamped in red "MASTER COPY" 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\

ADENOVIRUS QUANTITATIVE PCR BIO-RAD CFX96

Introduction: ................................................................................................................................................ 2

Specimen Collection, Transport & Storage ................................................................................................ 2

Materials, Equipments and Facilities: ......................................................................................................... 2

Specimen Processing .................................................................................................................................. 3

Procedure: ................................................................................................................................................... 4

General Precautions: .............................................................................................................................4

Build Worklist .......................................................................................................................................4

PCR Set-up: ............................................................................................................................................5

To be loaded by epMotion .....................................................................................................................7

To be loaded by manual pipetting; .......................................................................................................7

BIORAD Detection Area: ......................................................................................................................7

Import worklist ....................................................................................................................................10

Analysis .................................................................................................................................................15

Interpretation: ............................................................................................................................................ 19

Reporting ................................................................................................................................................. 19

Cleaning .................................................................................................................................................... 21

Quality Control ......................................................................................................................................... 22

Related Documents ................................................................................................................................... 23

References: ................................................................................................................................................ 23

Record of Edited Revisions ....................................................................................................................... 24


Quality Manual


Page



CFX96 Prepared by QA Committee

Issued by: Laboratory Manager Revision Date: 4/12/2021

Approved by Laboratory Director:

Microbiologist-in-Chief

Annual Review Date: 4/12/2023

Uncontrolled When Printed




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

Introduction:

Adenovirus is a double stranded non-enveloped DNA virus in the family Adenoviridae first isolated in

1950 from adenoid tissue. Adenoviruses is classified into 7 species A to G with several serotypes;

causing a wide range of illnesses from colds, diarrhea, eye infections to neurological diseases. Recently,

Adenovirus has been seen to cause severe infections in transplant recipients resulting in graft loss.

Adenovirus PCR is a qualitative real-time PCR, used for the detection of Adenovirus in sterile body

fluids, including plasma.

Specimen Collection, Transport & Storage

Fluids collected in a sterile container, store at C after collection, if processed within 24 hours; store at

-20oC if processing >24 hours.

EDTA blood: plasma should be removed from red cells 4-6 hours after collection. Centrifuge EDTA

blood at ≥10,000 RCF (Relative Centrifugal Force), and remove plasma 4-6 hours after collection. Store

plasma at C if processed within 24 hours; store at -20

oC if processing >24 hours.

Materials, Equipments and Facilities:

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

Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8)

BIO-RAD CFX96 Deep WellTM Real-Time System

BIO-RAD Hard-Shel®PCR Plates 96-Well WHT/CLR

BIO-RAD Microseal ®‘B’ seal Seals

BIO-RAD Optical Flat 8-Cap Strips for 0.2ml tube strips

BIO-RAD Low-Profile PCR Tubes 8-tube strip, white

96-Well Loading Block (pre-cooled to -20°C)

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

Reagents: Altona Adenovirus PCR Kit 1.0: MasterA, MasterB, DNA Internal Control,

Adenovirus QS3 as positive control and PCR grade water as a negative control.

External Controls: Adenovirus Externa control and a known negative external control to be run in

every new shipment.


Quality Manual


Page



CFX96




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

Specimen Processing

Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

https://eportal.mountsinai.ca/MSHPresentations/public/paradigm/D0024022.pdf


Quality Manual


Page



CFX96





Procedure:

General Precautions:

There must be separate PCR work areas:

Clean room

Specimen preparation room

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 thoroughly at room temperature.

PCR work areas (Clean Room and Specimen Preparation Area) benchtops and equipment after each

shift.

Build Worklist

Open the worklist file according to the following path:

T:Microbiology>Virology>Bio Rad CFX96 PCR>Worklist

Mastcopy>HZ_CMV_PARVO_ADENO Worklist

Password window pops up, click Read only button

Scan the samples’ information


Quality Manual


Page



CFX96





File > Save as CSV at

T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist

File name: Adeno yyyy.mm.dd.run No. eg.Adeno_2016.01.16

Save as type: CSV (Comma delimited)

Note:Worklist can only be imported as CSV type

Click Save button

Click OK

The following window pops up, click Yes button

Close current excel file without any savings.

PCR Set-up:

Prepare eluate samples including controls in order according to the Adenovirus worksheet.


Quality Manual


Page



CFX96





In the Clean Room:

Change into dedicated clean room gown and gloves, work in Biological Safety Cabinet.

Remove the required vials from -20oC freezer to thaw at room temperature the required number of vials’

of Adenovirus Master A, and Adenovirus Master B (12 reactions/vial) for your Adenovirus PCR run.

Prepare Adenovirus Master Mix in 1.5mL conical (sarstedt) microtube;

Number of Test Samples + 2 Controls (Adenovirus QS3&NC) + one extra

Mix gently, do not vortex. Make only enough master mix for the tests you are running. After

Master A has been added together to Master B it cannot be frozen again.

.

No. of Test

reactions

Adenovirus Master Mix

Adenovirus

Master A (µl)

Adenovirus

Master B (µl)

1 5 15

2 10 30

3 15 45

4 20 60

5 25 75

6 30 90

7 35 105

8 40 120

9 45 135

10 50 150

11 55 165

12 60 180

Number of Reactions 1

Adenovirus Master A 5

Adenovirus Master B 15

Volume of Master Mix 20

Sample/Control Volume 10


Quality Manual


Page



CFX96





To be loaded by epMotion

See Pipetting by epMotion Manualfor further loading and programming instructions.

To be loaded by manual pipetting;

Place 96-well plate onto pre-cooled block

Pipette 20 uL prepared Adenovirus Master Mix into each reaction well;

Pipette 1.0 uL of internal control into the wells designated for the positive and negative

controls

Pipette 10 uL PCR grade Water into the well designated for negative control.

In the Specimen Processing Area:

Pipette 10uL of each sample, QS, Negative control into reaction wells according to the plate

map. Mix by pipetting up and down 3 times into the Master Mix.

Seal the plate after pipetting is completed.

Load plate into the centrifuge carrier and spin at until speed hits 3000 rpm, then hit stop

Check reaction wells before loading into CFX96 Deep Well Real-Time System: ensuring the

liquid levels are at the same height and there are no bubbles at the bottom of each reaction well.

BIORAD Detection Area:

Double click Bio-Rad CFX Manager icon

Startup Wizard window pops up



Quality Manual


Page



CFX96





Make sure CFX96 Deep Well is selected

Under Select run type, click User-defined button

Run Set up window pops up, including three tabs-Protocol, Plate, and Start Run

Click Open Lid button to open the lid

Load sealed plate to the block

Click Close Lid button to close the lid

WARNING! Do NOT manually close the motorized lid

Under Protocol tab, select Altona DNA PCR.prcl from Express Load pull-down menu

Open and

Close

Lid

button

Express

Load pull-

down

menu


Quality Manual


Page



CFX96





Click Plate tab on the top or click Next button on the bottom right

side to load the plate profile

Select hz_cmv_parvo_adeno.pltd plate profile from the Express Load pull-down menu

Click Start Run tab on the top or click Next button on the

bottom right side


Quality Manual


Page



CFX96





Confirm the following information:

Protocol: Altona DNA PCR.prcl

Plate: Altona hz_cmv_parvo_adeno.pltd

Scan Mode: All Channels

If any information above needs to be edited, click Prev button

If all information is correct, click Start Run button

Save Optical Data File [CT014845] window pops up

Change “admin” to “Adeno”

Save the file in the designated folder:

T:Microbiology>Virology>Bio Rad CFX96 PCR>Save Run

Click Save button

Import worklist

Click Realtime Status tab

Select View/Edit Plate… from Plate Setup pull-down menu

Plate Editor window pops up

Plate Setup

pull-down

menu


Quality Manual


Page



CFX96





Click Spreadsheet View/Importer button

Plate Spreadsheet View window pops up

Select HEX from Fluors List pull-down menu


Quality Manual


Page



CFX96





Click Import button

Select Import File window pops up

T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist

Select appropriate worklist and click Open, all samples and controls information are imported

Click OK button

Select all wells by clicking top left side corner


Quality Manual


Page



CFX96





Type IC as target Name for HEX channel

Hit Enter key on the key board

Click Well groups button

Well Groups Manager window pops up

Click Add button

Select wells including control wells for Adeno detection (selected wells should be highlighted)

Click top left

side corner to

select all

wells Type IC and

hit Enter key


Quality Manual


Page



CFX96





Change the name Group 1 to Adeno

Click OK button

Click Ok button from Plate Editor window


Quality Manual


Page



CFX96





Click Yes button from the popping up window

Click Time Status tab from the Run Details window

Analysis

Choose Adeno from Well Groups pull-down menu

Change the channel names from Fluorophore to Target


Quality Manual


Page



CFX96





Set the threshold line for each target channel (Adeno & IC). Click off the ic leaving adeno on.

Drag the threshold over all the flat (negative) reaction. Repeat on the ic channel.

Check and mark on the worksheet the result of each sample and controls.

A positive PCR is observed if there is a rise or amplification in the Target channel e.g.

FAM (Adeno) and HEX (IC). The graph should be exponential and sigmoidal in shape.

Conversely a negative PCR is a flat line or no signal.

Click Export

Select Custom Export…

Click Export button


Quality Manual


Page



CFX96





Save as Window pops up

Change the File name according to the target and save in the following folder:

T:\microbiology\Virology\Bio Rad CFX96 PCR\Exported Excel Results\

Click Save button

The final results with Ct values are exported in Excel sheet

Change Starting Quantity to Scientific Notation

Print the excel sheet and attach it to the Adeno worksheet

Close excel

Click OK button


Quality Manual


Page



CFX96





Click Close button

Close CFX96 Deep Well Real-Time Systim

Shut down computer

Shut down Biorad CFX 96 Thermocycler.


Quality Manual


Page



CFX96





Interpretation:

Sample ID FAM (Adenovirus) HEX (IC) Interpretation

A Positive Negative POSITIVE for

Adenovirus*

B Negative Positive NEGATIVE for

Adenovirus

C Negative Negative PCR Inhibition:

Re-extract and repeat

PCR from extraction

* Negative Internal Control readings in HEX (IC) may occur in strongly positive FAM channels. High

Adenovirus viral loads in the sample often deplete the amplification/detection materials (eg.

nucleotides), leading to reduced or absent Internal Control signals.

Reporting

NEGATIVE RESULTS:

Negative for Adenovirus.

Under media on the back of the LIS workcard. PCADE: F6 to add run date

Report on the front of the LIS workcard from the keypad select: }ADE-

Negative for Adenovirus.

This is a research test.

Adenovirus PCR Kit, Altona Diagnostics GmbH.

Ctrl-F to finalize the report.

POSITIVE RESULTS:.

A. Non-Sterile sites:

Positive for Adenovirus

Date the media on the back of the LIS workcard. PCADE: F6 to add run date

Enter on the media line the cross point (ct) value: Ct=

Report the virus as an isolate in the F7 window.

Isolate #: 1 Org.ID. 21ade

Open the Isolate Comment window F8.


Quality Manual


Page



CFX96





Go to the Virology keypad, type “V”. Select from the keypad:

\ADE+

DETECTED by PCR,


Adenovirus PCR Kit, Altona Diagnostics

Verify the result.


B. Sterile sites:

PRELIMINARY Positive for Adenovirus

Date the media on the back of the LIS workcard. PCADE: F6 to add run date

Enter on the media line the cross point (ct) value: Ct=

Report the virus as an isolate in the F7 window.

Isolate #: 1 Org.ID. 21ade

Open the Isolate Comment window F8.

Go to the Virology keypad, type “V”. Select from the keypad:

\ADE+

DETECTED by PCR, confirmation to follow



Verify the result.

Ctrl-P to prelim the report. Call reports to ward/doctor.

Order media PRADE for repeat and repeat the sample from extraction to amplification.

If Repeat is positive:

CONFIRMED Positive Adenovirus. Date the media on the back of the LIS workcard. PRADE: F6 to add run date

Enter on the media line PRADE the cross point of the repeat (ct) value: Ct=

Open the Isolate Comment window F8, change the previous report “confirmation to follow” to

“Confirmed” e.g.

\ADE+ DETECTED by PCR, confirmed.



Verify the result.


If repeat is negative :


Quality Manual


Page



CFX96





NOT CONFIRMED for Adenovirus. Date the media PRADE on the back of the LIS workcard. PRADE: F6 to add run date

Isolate #: 1 must be suppressed, change to the corresponding alphabet letter eg. 1(A), 2(B)

In front of the LIS workcard type the following:

UPDATED REPORT: Previously reported Adenovirus not confirmed.

Report in front of the LIS workcard:


Adenovirus PCR Kit, Altona Diagnostics GmbH

Ctrl-F to finalize the report. Call Updated reports to ward/doctor.

Calling Results:

Adenovirus DETECTED from sterile body fluids, tissues, and eye fluids/swabs should be called to

ward and/or doctor.

Cleaning

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

Wash racks.

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.


Quality Manual


Page



CFX96





Quality Control

Reagent QCs:

An External Control (external to Altona Diagnostics) is used to monitor the isolation,

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

by the manufacturer.

External control positive and negative for Adenovirus should be extracted on easyMag and run at

with each new lot or shipment of reagent.

Daily QCs: Every Run

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

PCR inhibition.

A Positive Control (QS3) is included and should show a positive signal (amplification) in FAM

Channel (Adenovirus).

A Negative Control (water) is included and shows a negative reading in FAM Channel (VZV),

Texas Red Channel (HSV-1), and Cy5 (HSV-2).

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).


Quality Manual


Page



CFX96





Related Documents

Virology Accessioning Manual

Biorad Worksheet

Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

Pipetting by epMOTION Manual

Qualitative PCR External Control Log T:\microbiology\Virology\QC

statistics\EXTERNAL QC and

INVENTORY Logs\Qualitative

PCR EXTERNAL QC.xls

References:

Altona Adenovirus PCR Kit v1.0 Instructions, Altona Diagnostics






Quality Manual


Page



CFX96





Record of Edited Revisions

Manual Section Name: Adenovirus PCR Bio-Rad CFX96

Page Number / Item Date of Revision Signature of

Approval

Manual Transferred from Molecular Diagnostics Manual

Policy # MI/MD/v51 archived 2015.12.02

December 2, 2015 Dr. T. Mazzulli

Added Interpretation Section March 17, 2016 Dr. T. Mazzulli

Annual Review

Updated MSH logo in header.

Updated External QC and Daily QC section:

Added on each new lot or shipment

Updated Threshold Instructions:

Set the threshold line for each target channel (Adeno &

IC). Click off the ic leaving adeno on. Drag the threshold

over all the flat (negative) reaction. Repeat on the ic

channel.

Updated file paths to acces throughout manual

Update related link to new Qualitative PCR excel for QC

and inventory logging

August 24, 2016 Dr. T. Mazzulli

Modifed centrifugation time of spinning from 2 minutes

to when centrifuge reaches 3000rpm.

Moved build worklist section to beginning of procedure.

Updated reporting of sterile fluids and non-sterile

specimens

Added note for positive samples with negative internal

control in interpretation table.

December 9, 2016 Dr. T. Mazzulli

Annual Review September 15, 2017 Dr. T. Mazzulli


Annual Review October 02, 2019 Dr. T. Mazzulli

Minor format change February 20, 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:

Minor formatting change April 11, 2021 Jessica Bourke