S8-044-A1 GA-map Dysbiosis Test Installation Guide
- June 17, 2024
- GA
Table of Contents
INSTALLATION GUIDE
for GA-map® Dysbiosis Test Lx v2
OVERVIEW
This guide provides the information required to prepare a laboratory for performing the GA-map® Dysbiosis Test Lx v2 assay. Requirements for instruments, materials, and reagents are described.
INSTALLATION CHECK LIST
The following installation checklist summarizes the elements that must be completed during the installation phase. Mark each item as it is completed. Once these prerequisites have been met, please contact Genetic Analysis AS to schedule the training.
- Instruments and consumable items for Step 1 Genomic DNA extraction (see tables on page 4-6) must be acquired
- Instruments and consumable items for Step 2 Amplification of the bacterial 16S rRNA gene – Step 6 Hybridization and Signal Detection (see tables on page 6-8) must be acquired
- All instruments, including the Luminex detection system, must be installed and ready for use
- Operators must have been trained in use and maintenance of the Luminex detection system
RECOMMENDED SAFETY EQUIPMENT
**** Fecal samples should be treated as potentially infectious material until inactivation and require the use of BSL-2 grade laboratory equipment and precautions. This involves the use of appropriate PPE, biological safety cabinet, proper waste disposal and risk-minimizing routines for sample handling. Appropriate skin and eye protection should be worn during use of the DNA isolation kit (mag™ maxi, LGC genomics). For all other steps of the assay, suitable lab coat and disposable gloves should be used.
COMPUTER REQUIREMENTS
The GA-map® Analyzer is a cloud-based software compatible with most web browsers. See the GA-map® Analyzer Manual for details.
LABORATORY REQUIREMENTS
The GA-map® Dysbiosis Test Lx v2 requires a typical molecular laboratory set-
up. It is recommended that the laboratory contains, at a minimum, dedicated
pre-PCR, template-free, and PCR/post-PCR zones. The zones should be separated
to prevent contamination.
The table below contains an overview of the different laboratory procedures
that are performed in the individual zones, in addition to instruments and
equipment needed in the different workstations. Please refer to the Equipment,
materials, and reagents required section for equipment specifications. To
equip the lab with appropriate pipettes, please refer to the IFU for volumes
to be pipetted.
| Zone | Lab procedures | Workstation |
|---|---|---|
| Pre-PCR | Sample preparation and gDNA extraction. | |
| Addition of template for the amplification step. | Biological safety cabinet |
equipped with:
• Vortex mixer
• Decapper for Lysing Matrix-E tubes
• Appropriate single- and 8-channel pipettes w/ wide orifice tips
Open bench equipped with:
• Bead beater
• Plate centrifuge for Lysing Matrix-E tubes
• Water bath
• DNA extraction robot
• Vortex mixer
• Microcentrifuge
• Dispenser pipette w/ tips
• Single- and 8-channel pipettes with tips
• Ice or cooling elements for other reagents and plate
Template- free| Mastermix preparation for the amplification, clean-up, End-
Labeling, and Hybridization steps.| PCR cabinet with available equipment:
• Vortex mixer
• Microcentrifuge
• Dispenser pipette w/ tips
• Single channel pipettes w/ tips
• Freezing block for enzymes
• Ice or cooling elements for reagents and plate
PCR/Post-PCR| Amplification reaction.
The quantification step.
Addition of template for the End-labeling step and the End-labeling reaction.
The Hybridization and signal detection step.| Open bench equipped with:
• Plate centrifuge for quick spin
• Thermal cycler
• DNA quantification system
• Ring magnet plate
• Luminex detection platform
• Vortex mixer
• Microcentrifuge
• Dispenser pipette w/ tips
• Single- and 8-channel pipettes w/ tips
• Ice or cooling elements for reagents and plate
• Freezing block for plates
EQUIPMENT, MATERIALS, AND REAGENTS REQUIRED
The lists below describe the required equipment, materials, and reagents for
the GA-map® Dysbiosis Test Lx v2.
All items are required, unless otherwise specified. Genetic Analysis must be
informed of any deviations from these requirements and a separate validation
might be necessary.
The quantity listed for all equipment is based on the set-up described in the
Laboratory Requirements section above and describes the minimum recommended
quantity. It should be noted that different laboratory set-ups (e.g. with
additional zones) might affect the recommended quantity.
FOR STEP 1 – GENOMIC DNA EXTRACTION
List of equipment
| Equipment type| Specifications/ requirement| Alternative or
recommended option| Quantity
---|---|---|---|---
| Decapper for Lysing Matrix-E tubes| Capacity: 8-channel capper/decapper for
matrix tubes| 8-Channel Screw Cap Decapper (4105MAT), Thermo Scientific| 1
AlteCap™ Switch (404000) with Cassette 12/96 – Internal thread Matrix screw
caps (404014), AltemisLab
| Bead beater| FastPrep-96™ Homogenizer w/96-well plate insert (116010500),
MP Biomedicals| No validated alternatives| 1
| Plate centrifuge for Lysing
Matrix-E tubes
| Capacity: Lysing Matrix-E 96 well rack with tubes (6 cm height) Force: 1300
rcf| Eppendorf™ Centrifuge Centrifuge 5804/5810 w/ rotor for deepwell plates,
Eppendorf| 1
| Water bath| Capacity: deep well 96 plate Temperature: 65°C| Any| 1
| DNA
extraction robot
| KingFisher Flex – 96 deep well head (540 0630) w/magnetic micro-plate
Separator, installed with KF Flex 96 KF heating block (2407 5420), Thermo
Scientific| No validated alternatives| 1
| Vortex mixer| Speed: ~2800 rpm| Any| 2
| Micro Centrifuge| Capacity: 1.5ml/ 2ml tubes (spin down only)| Any| 1
| Dispenser pipette w/tips| Volumes to be dispensed: 20, 200, 250, 270 and
720 µl| Multipette®E3/E3x (4987000371/4987000380),
Eppendorf
| 1
| Pipettes single channel w/tips| Volumes: see IFU
Wide orifice tips recommended for pipetting of fecal samples prior to gDNA
extraction| Any| Depends on volume range
| Pipettes 8- channel w/tips| Volumes: see IFU
Wide orifice tips recommended for pipetting of fecal samples prior to gDNA
extraction| Any| Depends on volume range
| Ice or cooling blocks| For keeping reagents/sample intermediates cold
during handling| Any| NA
List of materials
| Material| Specifications/ requirement| Alternative or
recommended option
---|---|---|---
| Lysing Matrix-E tubes| Lysing Matrix E, 96-tube rack, barcoded tubes, 1
Rack (116984001B), (note: individual Matrix tubes with screw cap), MP
Biomedicals| No validated alternatives
| Tube for Lysis mix| Volume: 5, 15 or 50 ml| Any
| Deep well plate for DNA extraction robot| KingFisher Deepwell 96 Plate, V-
bottom, (95040450), Thermo Scientific| 96 Well Plate SW 2ml with V- Bottom PP
(BAKR43001-0504), VWR
| Deep well Tip Combs for DNA extraction robot| KingFisher 96 tip comb for DW
magnets, (97002534), Thermo Scientific| 96 Tip Comb PP (BAKR43001-0505), VWR
| Elution plate for DNA extraction robot| KingFisher 96 KF microplate
(200μL), (97002540), Thermo
Scientific
| Well Plate 96 SW 0.2ml with V- Bottom PP (BAKR43001-0506), VWR
| Adhesive PCR plate seal| Suitable for deep well plate for DNA extraction
robot| Adhesive PCR Plate Seals (AB0558), Thermo Scientific
| Microtiter sealing tape| Suitable for plates for DNA extraction robot|
Adhesive Plate Seals (AB0580), Thermo Scientific
| Microtiter plate w/seal for gDNA dilution| Capacity: 96-well, ≥250µl| Any
| Reagent reservoir| ≥25 ml| Any
List of reagents
| Reagent| Specifications/ requirement| Alternative or
recommended option
---|---|---|---
| Extraction control positive (optional)| Fecal sample of known quality| Any
| DNA extraction reagent kit| mag™ maxi DNA purification kit, 288 tests
(NAP40430), LGC Genomics
Note! Protease must be stored at -20°C upon reception| No validated
alternatives
| Ethanol (for extraction kit)| 96-100% ethanol
≥500ml| Any
| Acetone (for extraction kit)| ≥99% acetone
≥350ml| Any
| Water for dilution of gDNA and as PCR ctrl neg.| Sterile, Nuclease-free
water
≥500ml| Any
FOR STEP 2 – AMPLIFICATION OF THE BACTERIAL 16S RRNA GENE TO STEP 6
–HYBRIDIZATION AND SIGNAL DETECTION
List of equipment
| Equipment type| Specifications/ requirement| Alternative or
recommended option| Used in assay steps| Quantity
---|---|---|---|---|---
| Plate centrifuge| Capacity: 96-well plate (quick spin down only)| Any| Step
2
Step 3
Step 4
Step 5
Step 6| 1
| Thermal cycler| VeritiPro™ 96-Well Thermal Cycler (A48141), Applied
Biosystems| Veriti™ 96-Well Thermal Cycler (4375786), Applied Biosystems| Step
2
Step 4
Step 5
Step 6| 1
T100™ Thermal Cycler (186- 1096), Bio-Rad
| DNA
quantification system
| FLUOstar OMEGA Microplate Reader with filter-based absorbance (415-103) w/
appropriate filters for the quantification assay, BMG LabTech| Qubit®
Fluorometer 3.0 (Q33216) or 4.0 (Q33238) or Flex (Q33327), Invitrogen| Step 3|
1
| Ring Magnet Plate| Capacity: 96-well Hybridization plate, 100 µL| 96-Well
Ring Magnet Plate (S380), Permagen| Step 6| 1
| Luminex detection platform| Luminex® 200™ system with xPONENT® version 4.2
or higher (LX200- XPON-IVD/RUO), Luminex| MAGPIX® system or NxTAG®‑Enabled
MAGPIX® system with xPONENT® version 4.2 or higher (MAGPIX-XPON4.1-CEIVD),
Luminex| Step 6| 1
| Vortex mixer| Speed: ~2800 rpm| Any| Step 2
Step 3
Step 4
Step 5
Step 6| 3
(including one of the two listed in Step 1)
| Micro Centrifuge| Capacity: 2 ml tubes
(spin down only)| Any| Step 2
Step 3
Step 4
Step 5
Step 6| 3
(including the one listed in Step 1)
| Dispenser pipette w/tips| Volumes to be dispensed: 20 µl and 40 µl|
Multipette®E3/E3x (4987000371/4987000380), Eppendorf| Step 2
Step 5
Step 6| 2
| Pipettes single channel w/tips| Volumes: see IFU| Any| Step 2
Step 3
Step 4
Step 5
Step 6| Depends on volume range
| Pipettes 8- channel w/tips| Volumes: see IFU| Any| Step 2
Step 3
Step 4
Step 5
Step 6| Depends on volume range
| Ice or cooling blocks| For keeping reagents/sample intermediates cold
during handling| Any| Step 2
Step 3
Step 4
Step 5
Step 6| NA
| Freezing block for tubes| Capacity: 1.5/ 2 ml reagent tubes
For use during handling of enzymes| Benchtop cooler (5115- 0012), Thermo
Scientific| Step 2
Step 4
Step 5| 1
| Freezing block for plates| Capacity: 96-well microtiter plate
For use during End- labeling| PCR-Cooler 0.2 mL (3881000031), Eppendorf| Step
5| 1
*Additional items for use and maintenance are required: kits for calibration and performance verification, sheath fluid or drive fluid, and an ultrasonic bath (40-60 kHz)
List of materials
| Material| Specifications/ requirement| Alternative or
recommended option| Used in assay steps
---|---|---|---|---
| Microcentrifuge tubes for mixing of reagents| Volume: 1.5, 2 and 5 ml|
Safe-Lock tubes, Eppendorf| Step 2
Step 4
Step 5
Step 6
| Microtiter plate for PCR/End- labeling| Capacity: 96-well PCR grade|
96-Well Semi-skirted Flat Deck PCR Plates (AB1400), Thermo Scientific| Step 2
Step 5
| 8-cap sealing strips| Suitable for microtiter plate| Flat PCR Caps, strips
of 8 (AB0784), Thermo Scientific| Step 2
Step 5
| Microtiter sealing tape| Suitable for microtiter plate| Adhesive Plate
Seals (AB0580), Thermo Scientific| Step 2
Step 5
| Tubes/plates for DNA quantification| Suitable for the selected DNA
quantification system| For use with plate reader: Nunc™ 96-Well Microplate,
Black (237108), Thermo Scientific| Step 3
For use with Qubit® 3.0/4.0 Fluorometer: Qubit™ Assay Tubes (Q32856),
Invitrogen
For use with Qubit® Flex Fluorometer: Qubit™ Flex Assay Tube Strips (Q33252),
Invitrogen
| Hybridization plate| Thermowell™ 96-Well Polycarbonate PCR Microplates,
Model P (6509), Corning| 96-well Twin.tec™ PCR Plates, Unskirted, Divisible
(0030133358), Eppendorf| Step 6
| Sealing film for Hybridization plate| Microseal® ‘A’ (MSA5001), Bio-Rad| No
validated alternatives| Step 6
| Pierceable foil for Hybridization plate**| Pierceable foil to cover 96-well
plate| Any| Step 6
| Reagent reservoir| ≥25 ml| Any| Step 4
Step 6
Only applicable if using the NxTAG®-enabled MAGPIX® detection platform
List of reagents**
| Reagent| Specifications/ requirement| Alternative or
recommended option| Used in assay steps
---|---|---|---|---
| Water for PCR control neg.| Sterile, Nuclease-free water, same as used for
dilution of gDNA| Any| Step 2
| Assay kit for DNA quantification| For use with plate reader : Quant-iT™
1X dsDNA HS Assay (Q33232), ThermoFisher| Quant-iT™ PicoGreen™ dsDNA Assay Kit
(P11496), ThermoFisher| Step 3
For use with Qubit® Fluorometer : Qubit™ 1X dsDNA HS assay kit (Q33231),
ThermoFisher| Qubit™ dsDNA HS assay kit (Q32854), ThermoFisher
TRAINING PLAN
Training will be scheduled following system set-up. Genetic Analysis will
train users in performing the process of GA-map® Dysbiosis Test Lx v2 from
fecal gDNA extraction (or from PCR, if the extraction method differs from that
described in the IFU) to generation of the reports. Basic instrument operation
and use of the software will be included in the training. Those being trained
will be required to have a basic knowledge of Microsoft Windows and use of
general laboratory equipment and tools.
Operators must have been trained in use and maintenance of the Luminex system
prior to the tech transfer.
CONTACT INFO
We are happy to help you with your inquiries.
Technical Support: support@genetic-analysis.com
Registered names, trademarks, etc. used in this document, even when not
specifically marked as such, are not to be considered unprotected by law. All
trademarks and registered trademarks mentioned herein are the property of
their respective owners.
Genetic Analysis AS | Ulvenveien 80 | 0581 OSLO | Norway
www.genetic-analysis.com
Phone: +47 48 32 16 10
E-mail: info@genetic-analysis.com
© 2021-2023 Genetic Analysis, all rights reserved.
S8-044 A1 v8 Appendix 6