Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

Brand
Thermo Fisher Scientific
Model
VetMAX BVDV 4ALL Kit
Type
Operating instructions
For Laboratory Use.
VetMAX™BVDV4ALLKit
SUPPLEMENTAL INSTRUCTIONS
Bovine Viral Diarrhea Virus viral RNA purification protocols
compatible with the VetMAX™BVDV4ALLKit
for use with:
Species: Bovine and small ruminants (sheep, goats)
Matrices: Blood, serum, and ear biopsies
Test type: Individual or pooled (according to the type of specimen)
Catalog NumberBVD4ALL
Document Part Number100021235
Publication NumberMAN0008962
Revision G.0
Laboratoire Service International (LSI) | 6 Allée des Ecureuils – Parc Tertiaire du Bois-Dieu | 69380 Lissieu – France
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No.MAN0008962
Revision Date Description
G.0 28 September 2022
•A new chapter for liquid and wet ear biopsies was created (see Chapter 4, “RNA extraction from
liquid and wet ear biopsies”).
•The important note for ocial testing incubation time for wet ear biopsies was added (see “Prepare
the ear biopsy lysate” on page46).
•The protocols for the MagVet™ Universal Isolation Kit were removed from the document.
•Minor wording updates were completed for consistency with related documents.
F.0 30 January 2020
•Added the One‑Step Heating Workflow that only requires a single heating step for the rapid lysis of
ear biopsies (see Chapter5, “Rapid lysis for ear biopsies”).
•Updated the list of required materials that are not supplied with the kit.
E.0 7 November 2018
•Added instructions for nucleic acid purification with the MagMAX™ CORE Nucleic Acid Purification
Kit.
•Added Appendix A, “Blood storage guidelines applicable to France”.
D.0 20 June 2017 Updated to the current document template, with associated updates to the warranty, trademarks, and
logos.
C.0 16 January 2015
•Corrected NucleoSpin™ RNA kit name.
•In step 10 of “Extraction with the QIAamp™ Viral RNA Mini Kit”, added instructions to place the
column in a new collection tube.
B.0 22 April 2014 Added instructions for use of 5 - IPC BVDV 4ALL.
A.0 13 December 2013 New document.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Eppendorf™
is a trademark of Eppendorf AG. NucleoSpin™ is a trademark of MACHEREY‑NAGEL. QIAGEN™, QIAamp™, and RNeasy™ are
trademarks of QIAGEN GmbH.
©2022 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER1Overview .............................................................. 6
Sampleselection ................................................................ 6
Cell-free samples and wholeblood ............................................ 6
Cellsamples ............................................................... 7
Sample storage ................................................................. 7
Whole blood (in EDTAtubes) ................................................. 7
Serum ..................................................................... 7
Earbiopsies ................................................................ 7
Required materials notsupplied ................................................... 8
Recommended RNA extraction protocols ......................................... 10
■CHAPTER2RNA extraction from EDTA blood,serum ......................... 11
Preparation of samples before extraction .......................................... 11
Whole blood collected in EDTAtubes ........................................ 11
Serum for individualanalysis ................................................ 11
Pooled serum or whole bloodanalyses ....................................... 11
Guidelines for exogenous Internal Positive Control (IPC)use ......................... 12
Extraction with the MagMAX™ CORE Nucleic Acid PurificationKit .................... 12
Recommendedworkflows .................................................. 12
Workflow: Multisample Simple .............................................. 13
Workflow: Simple .......................................................... 13
Proceduralguidelines ...................................................... 14
Before first use of thekit .................................................... 14
Set up the processing plates (KingFisher™ Flex and MagMAX™
Express-96instruments) .................................................. 15
Prepare samples for processing using the Multisample SimpleWorkflow ......... 16
Prepare samples for processing using the SimpleWorkflow ..................... 17
Set up the processing plates or tube strips (KingFisher™ Duo Prime and
KingFisher™mLinstruments) .............................................. 19
Process samples on theinstrument .......................................... 19
Extraction with the MagMAX™‑96 Viral RNA IsolationKit ............................ 20
Notes .................................................................... 20
Before starting ............................................................ 20
Protocol .................................................................. 21
VetMAX™BVDV4ALLKitSupplemental Instructions 3
Extraction with the QIAamp™ Viral RNA MiniKit .................................... 22
Before starting ............................................................ 22
Protocol .................................................................. 22
Extraction with the NucleoSpin™ RNA Viruskit ..................................... 23
Before starting ............................................................ 23
Protocol .................................................................. 24
■CHAPTER3RNA extraction from dry earbiopsies ............................. 26
Extraction with the MagMAX™ CORE Nucleic Acid PurificationKit .................... 27
Workflow ................................................................. 27
Proceduralguidelines ...................................................... 28
Guidelines for sample storage ............................................... 29
Before first use of thekit .................................................... 29
Set up the processing plates (KingFisher™ Flex and MagMAX™
Express-96instruments) .................................................. 30
Prepare Lysis/Binding/ BeadMix ............................................. 30
Prepare the ear biopsy lysate ................................................ 30
Combine the lysate with PK (optional), then add Lysis/Binding/ BeadMix ......... 31
Set up the processing plates or tube strips (KingFisher™ Duo Prime and
KingFisher™mLinstruments) .............................................. 33
Process samples on theinstrument .......................................... 33
Preparation of samples before extraction with otherkits ............................. 34
Earbiopsies ............................................................... 34
Extraction with the MagMAX™-96 Viral RNA IsolationKit ............................ 34
Before starting ............................................................ 34
Protocol .................................................................. 35
Extraction with the RNeasy™ MiniKit ............................................. 36
Before starting ............................................................ 36
Protocol .................................................................. 37
Extraction with the NucleoSpin™ RNAkit .......................................... 38
Before starting ............................................................ 38
Protocol .................................................................. 38
■CHAPTER4RNA extraction from liquid and wet earbiopsies ................ 40
Extraction with the MagMAX™ CORE Nucleic Acid PurificationKit .................... 40
Recommended workflows andscripts ........................................ 40
Multisample Simple Workflow: Liquid earbiopsy ............................... 41
Express Workflow: Wet earbiopsy ........................................... 43
Proceduralguidelines ...................................................... 43
Before first use of thekit .................................................... 44
Prepare samples for processing using the Multisample SimpleWorkflow ......... 45
Prepare samples for processing using the ExpressWorkflow .................... 49
Contents
4VetMAX™BVDV4ALLKitSupplemental Instructions
■CHAPTER5Rapid lysis for earbiopsies ........................................ 53
Preparation of samples before extraction .......................................... 53
Earbiopsies ............................................................... 53
Rapid lysis with the VetMAX™ Ear Notch Fast LysisKit .............................. 53
Recommendedworkflows .................................................. 53
Before youbegin .......................................................... 53
Prepare Fast Lysis Solution(FLS) ............................................ 53
Lyse samples using the One-Step HeatingWorkflow ........................... 54
Lyse samples using the Two-Step HeatingWorkflow ........................... 54
Analyze thesamples ....................................................... 55
■APPENDIXABlood storage guidelines applicable to France ................. 56
■Documentation and support ....................................................... 57
Customer and technical support ................................................. 57
Limited product warranty ........................................................ 57
Contents
VetMAX™BVDV4ALLKitSupplemental Instructions 5
Overview
IMPORTANT! In France, follow Appendix A, “Blood storage guidelines applicable to France”.
Species Isolation of nucleic acid from
matrices Test type
Bovine
Small ruminants (sheep, goats)
Blood
Serum
Ear biopsies
Individual or pooled
(according to the type of specimen)
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from
thermofisher.com/support.
WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this
product's page at thermofisher.com. Wear appropriate protective eyewear, clothing, and gloves.
Sample selection
Cell-free samples and whole blood
Animal Sample matrix Type of analysis Sampling equipment
Calf less than 1 month old Whole blood Individual[1] EDTA tube
Serum Individual[1] Dry tube with or without separator
Cattle more than 1 month old Whole blood Individual or pooled EDTA tube
Serum Individual or pooled Dry tube with or without separator
[1] It is recommended to test this sample type individually.
Refer to the national, regional, or local regulations in eect for the size of the pools.
1
6VetMAX™BVDV4ALLKitSupplemental Instructions
Cell samples
Animal Sample matrix Type of analysis Sampling equipment
Calves Skin samples Individual or pooled Ear biopsies
Refer to the national, regional, or local regulations in eect for the size of the pools.
Sample storage
The quality of the samples must be assured before starting the analytical process.
Whole blood (in EDTA tubes)
Collect blood in EDTA tubes. Following collection, maintain at 2°C to 8°C until use and for a maximum
of 8 days after sampling. After use or after the 8‑day period, freeze below −16°C for storage up to 1 year
or below −70°C for storage longer than 1 year.
Note: In France, store blood samples according to Appendix A, “Blood storage guidelines applicable to
France”.
Serum
Collect sera in dry tubes. Following collection, maintain at 2°C to 8°C until use and for a maximum of 8
days after sampling. After use or after the 8-day period, freeze below −16°C for storage up to 1 year or
below −70°C for storage longer than 1 year.
Ear biopsies
Sample type Storage
Dry ear biopsy
After collection, maintain below −16°C until use. After use, freeze below
−16°C for storage up to 1 year or below −70°C for storage longer than
1 year.
Liquid and wet ear biopsy (in
Allflex™ buer)
After collection, maintain the Allflex™ tube containing the skin biopsy at
room temperature, protected from light, for up to 1month.
Store lysates for retesting as indicated:
•Store individual lysates at room temperature for up to 1month, or
below −16°C for long‑term storage.
•Store pooled lysates at room temperature for up to 1 week.
Chapter1Overview
Sample storage 1
VetMAX™BVDV4ALLKitSupplemental Instructions 7
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.
Table1Materials required for all sample preparation and extraction methods
Item Source
Equipment
Class II microbiological safety cabinet (MSC) MLS
Precision micropipettes (range of 1 µL to 1,000µL) with DNase/RNase‑free filter tips MLS
Laboratory mixer, vortex or equivalent MLS
Benchtop microcentrifuge capable of 8,000–15,000 × g (for 1.5‑mL and 2‑mL
microtubes) MLS
Reagents
RNase/DNase‑free water (for negative control samples (NCS)) MLS
Consumables
1.5‑mL DNase/RNase‑free microtubes MLS
Table2Additional materials required for automated magnetic bead extraction
Item Source
Instrument and equipment
One of the following instruments[1]:
•KingFisher™ Flex Purification System
•MagMAX™ Express‑96 Magnetic Particle Processor
•KingFisher™ Duo Prime Purification System
•KingFisher™ mL Food Protection Purification System
•MagMAX™ Express-24 Magnetic Particle Processor
Contact your local sales
oce.
Kits and reagents[1]
One of the following kits:
•MagMAX™ CORE Nucleic Acid Purification Kit
•MagMAX™-96 Viral RNA Isolation Kit
•A32700 or A32702
•AM1836
Isopropanol (100%)[2] MLS
[1] Depending on the extraction protocol used.
[2] Required for use with the MagMAX™-96 Viral RNA Isolation Kit only.
Chapter1Overview
Required materials not supplied
1
8VetMAX™BVDV4ALLKitSupplemental Instructions
Table3Additional materials required for manual extraction on silica columns (blood and serum samples
only)
Item Source
Kits and reagents
One of the following kits:
•QIAamp™ Viral RNA Mini Kit
•NucleoSpin™ RNA Virus kit
•QIAGEN™
•MACHEREY‑NAGEL
Table4Additional materials required for manual extraction on silica columns (ear biopsies only)
Item Source
Kits and reagents
One of the following kits:
•RNeasy™MiniKit
•NucleoSpin™RNA kit
•QIAGEN™
•MACHEREY‑NAGEL
Consumables
QIAshredder[1] QIAGEN™
[1] Required for use with the RNeasy™MiniKit only.
Table5Additional materials required for rapid lysis of ear biopsies
Item Source
Equipment
Incubator or heat block set to 95°C[1] MLS
Two Incubators or heat blocks set to 70°C and 90°C[2] MLS
Kits and reagents
VetMAX™ Ear Notch Fast Lysis Kit FLK
Consumables
Microtubes (compatible with the incubator or heat block that is used) MLS
[1] Required for the One‑Step Heating Workflow.
[2] Required for the Two‑Step Heating Workflow.
Chapter1Overview
Required materials not supplied 1
VetMAX™BVDV4ALLKitSupplemental Instructions 9
Recommended RNA extraction protocols
Recommended extraction kits
Whole blood, serum MagMAX™ CORE Nucleic Acid Purification Kit
MagMAX™-96 Viral RNA Isolation Kit
QIAamp™ Viral RNA Mini Kit
NucleoSpin™ RNA Virus kit
Ear biopsies MagMAX™ CORE Nucleic Acid Purification Kit
MagMAX™-96 Viral RNA Isolation Kit
RNeasy™MiniKit
NucleoSpin™RNA kit
Ear biopsies (rapid lysis) VetMAX™ Ear Notch Fast Lysis Kit
Chapter1Overview
Recommended RNA extraction protocols
1
10 VetMAX™BVDV4ALLKitSupplemental Instructions
RNA extraction from EDTA blood,
serum
■Preparation of samples before extraction ................................................ 11
■Guidelines for exogenous Internal Positive Control (IPC)use ............................... 12
■Extraction with the MagMAX™ CORE Nucleic Acid PurificationKit .......................... 12
■Extraction with the MagMAX™‑96 Viral RNA IsolationKit .................................. 20
■Extraction with the QIAamp™ Viral RNA MiniKit .......................................... 22
■Extraction with the NucleoSpin™ RNA Viruskit ........................................... 23
Preparation of samples before extraction
Whole blood collected in EDTA tubes
Whole blood collected in EDTA tubes is used directly, without preparation prior to extraction.
The extraction requires 50–200µL of blood.
Serum for individual analysis
Serum, issued from blood collected in dry tubes, is used directly, without preparation prior to
extraction.
The extraction requires 50–200µL of serum.
Pooled serum or whole blood analyses
The pools may be made from dierent matrices. However, never mix blood collected in heparin
tubes with another matrix.
Pool samples manually or by machine. To limit the number of retests in the case of positive pools, first
combine individual samples into sub-pools, then combine sub-pools into a larger pool.
To avoid contamination in the case of manual pooling, the pipette tip must be changed after the
addition of each individual sample and each constituent of the sample pool.
In the case of automatic pooling, the risk of contamination between wells by the machine must
be checked according to the manufacturer’s recommendations.
2
VetMAX™BVDV4ALLKitSupplemental Instructions 11
For each analyzed pool:
•If the result of the analysis is negative: all samples in the pool will be considered negative.
•If the result of the analysis is positive: test all sub-pools included in the pool or each sample
individually if no sub-pools were made.
Similarly, for each subsequent sub-pool:
•If the result of the analysis is negative: all samples in the sub-pool will be considered negative.
•If the result of the analysis is positive: test all samples included in the sub-pool individually.
Guidelines for exogenous Internal Positive Control (IPC) use
Serum samples require an exogenous IPC source (5 - IPC BVDV 4ALL) that is added to the lysis buer
during RNA extraction. The IPC target is already present in the blood samples, so addition of 5 - IPC
BVDV 4ALL is not required for these samples.
•For extraction series that include only serum samples, add 5 µL of 5 - IPC BVDV 4ALL to the lysis
buer during RNA extraction of the samples, BVDV positive control, and the negative extraction
control sample (NCS).
•For extraction series that include only blood samples, prepare lysis solution without 5 - IPC BVDV
4ALL for the blood samples, the BVDV positive control, and the NCS.
•For extraction series that include both serum and blood samples:
–Add 5 µL of 5 - IPC BVDV 4ALL to the lysis solution for all the serum samples, the BVDV
positive control, and the NCS.
–Prepare lysis solution without 5 - IPC BVDV 4ALL for the blood samples in the extraction
series.
Extraction with the MagMAX™ CORE Nucleic Acid
Purification Kit
Recommended workflows
Two workflow options are available for the extraction of blood and serum samples using the MagMAX™
CORE Nucleic Acid Purification Kit:
•Multisample Simple Workflow—Recommended for processing a combination of sample types.
•Simple Workflow—Recommended for processing blood and serum samples only.
Note: The Multisample Simple and Simple Workflows dier only in the order that the reagents are
added to the samples.
Chapter2RNA extraction from EDTA blood, serum
Guidelines for exogenous Internal Positive Control (IPC) use
2
12 VetMAX™BVDV4ALLKitSupplemental Instructions
Workflow: Multisample Simple
Start with serum or whole blood samples (individual or pooled)
▼
Follow the appropriate procedure based on your instrument:
KingFisher™ Flex or MagMAX™ Express-96 KingFisher™ Duo Prime or KingFisher™mL
Set up the processing plates (page15)
▼
Prepare samples for processing using the
Multisample Simple Workflow (page16)
Prepare Lysis/Binding/Bead Mix (page16)
Prepare the sample (page16)
Combine the sample with PK, then add Lysis/
Binding/Bead Mix (page16)
▼
Process samples on the instrument (page19)
Prepare samples for processing using the
Multisample Simple Workflow (page16)
Prepare Lysis/Binding/Bead Mix (page16)
Prepare the sample (page16)
Combine the sample with PK, then add Lysis/
Binding/Bead Mix (page16)
▼
Set up the processing plates (page19)
▼
Process samples on the instrument (page19)
Workflow: Simple
Start with serum or whole blood samples (individual or pooled)
▼
Follow the appropriate procedure based on your instrument:
KingFisher™ Flex or MagMAX™ Express-96 KingFisher™ Duo Prime or KingFisher™mL
Set up the processing plates (page15)
▼
Prepare samples for processing using the
Simple Workflow (page17)
Prepare Bead/PKMix (page17)
Prepare Lysis/Binding Solution (page17)
Prepare the sample (page18)
Combine the sample with Bead/PKMix, then add
Lysis/Binding Solution (page18)
▼
Process samples on the instrument (page19)
Prepare samples for processing using the Simple
Workflow (page17)
Prepare Bead/PKMix (page17)
Prepare Lysis/Binding Solution (page17)
Prepare the sample (page18)
Combine the sample with Bead/PKMix, then add
Lysis/Binding Solution (page18)
▼
Set up the processing plates (page19)
▼
Process samples on the instrument (page19)
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit 2
VetMAX™BVDV4ALLKitSupplemental Instructions 13
Procedural guidelines
•Before use, invert bottles of solutions and buers to ensure thorough mixing.
•Mix samples with reagents using a plate shaker or by pipetting up and down.
Note: Do not use a plate shaker with the tube strips required by the KingFisher™mL instrument.
•To prevent cross-contamination:
–Cover the plate or tube strip during the incubation and shaking steps, to prevent spill-over.
–Carefully pipet reagents and samples, to avoid splashing.
•To prevent nuclease contamination:
–Wear laboratory gloves during the procedures. Gloves protect you from the reagents, and they
protect the nucleic acid from nucleases that are present on skin.
–Use nucleic acid-free pipette tips to handle the reagents, and avoid putting used tips into the
reagent containers.
–Decontaminate lab benches and pipettes before you begin.
Before first use of the kit
(Optional) Determine the maximum plate shaker setting
If a plate shaker is used, determine the maximum setting:
1. Verify that the plate fits securely on your shaker.
2. Add 1 mL of water to each well of the plate, then cover with sealing foil.
3. Determine the maximum setting that you can use on your shaker without any of the water
splashing onto the sealing foil.
Download the script
The scripts for the MagMAX™ CORE Nucleic Acid Purification Kit are not pre-installed on the
instruments.
1. On the MagMAX™ CORE Nucleic Acid Purification Kit product web page (at thermofisher.com,
search by catalogue number), scroll to the Product Literature section.
2. Right‑click the appropriate file to download the latest version of the MagMAX_CORE script for your
instrument.
Table6Recommended scripts
Instrument Script name
KingFisher™ Flex MagMAX_CORE_Flex.bdz
KingFisher™96
MagMAX™ Express-96
MagMAX_CORE_KF-96.bdz
KingFisher™ Duo Prime MagMAX_CORE_DUO.bdz
KingFisher™mL MagMAX_CORE_mL_no_heat.bdz
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit
2
14 VetMAX™BVDV4ALLKitSupplemental Instructions
If required by your laboratory, use one of the following scripts, which do not heat the samples
during the elution step.
Table7Alternate scripts without heated elution step
Instrument Script name
KingFisher™ Flex MagMAX_CORE_Flex_no_heat.bdz
KingFisher™96
MagMAX™ Express-96
MagMAX_CORE_KF-96_no_heat.bdz
KingFisher™ Duo Prime MagMAX_CORE_DUO_no_heat.bdz
KingFisher™mL MagMAX_CORE_mL_no_heat.bdz
See your instrument user guide or contact Technical Support for instructions for installing the script.
Set up the processing plates (KingFisher™ Flex and MagMAX™ Express-96
instruments)
IMPORTANT! If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, do not set up
the processing plates or tube strips before preparing the samples.
1. Set up the processing plates.
Table8Plate setup: KingFisher™ Flex or MagMAX™ Express-96 instrument
Plate ID Plate
position[1] Plate type Reagent Volume per
well
Wash Plate 1 2 Deep Well MagMAX™ CORE Wash Solution 1 500µL
Wash Plate 2 3 Deep Well MagMAX™ CORE Wash Solution 2 500µL
Elution 4 Standard MagMAX™ CORE Elution Buer 90µL
Tip Comb 5 Standard Place a tip comb in the plate.
[1] Position on the instrument.
2. (Optional) To prevent evaporation and contamination, cover the prepared processing plates with
sealing foil until they are loaded into the instrument.
3. Proceed to the workflow that is appropriate for your laboratory.
•Multisample Simple Workflow—See “Prepare samples for processing using the Multisample
Simple Workflow” on page16.
•Simple Workflow—See “Prepare samples for processing using the Simple Workflow” on
page17.
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit 2
VetMAX™BVDV4ALLKitSupplemental Instructions 15
Prepare samples for processing using the Multisample Simple Workflow
Prepare Lysis/Binding/Bead Mix
1. Combine the following components, in the order indicated, for the required number of samples
plus 10% overage.
Component Volume per sample
MagMAX™ CORE Lysis Solution 350µL
MagMAX™ CORE Binding Solution 350 µL
MagMAX™ CORE Magnetic Beads 20µL
Total Lysis/Binding/Bead Mix (−IPC) 720µL
5-IPC BVDV 4ALL[1] 5 µL
Total Lysis/Binding/Bead Mix (+IPC) 725µL
[1] Add 5-IPC BVDV 4ALL only to serum samples, and to BVDV positive control and NCS in extraction series that include at least
one serum sample.
2. Mix by inverting the tube or bottle at least 10times.
Prepare the sample
Prepare samples and controls as described.
Sample type Action
Individual samples Proceed with 200µL of sample.
Pooled samples Proceed with 200µL of pooled samples.[1]
BVDV positive control Proceed with 50µL of 4c-EPC BVDV 4ALL.
NCS —
[1] For information on the preparation of samples before extraction, see “Pooled serum or whole blood analyses” on page11.
Combine the sample with PK, then add Lysis/Binding/Bead Mix
1. Add 10µL of MagMAX™ CORE Proteinase K to the required wells in the plate or tube strip.
2. Transfer each prepared sample or control to a well with MagMAX™ CORE Proteinase K.
3. Mix the sample with Proteinase K for 2 minutes at room temperature according to your mixing
method.
•Using a plate shaker: shake vigorously for 2 minutes (see “(Optional) Determine the maximum
plate shaker setting” on page14).
•By pipetting: pipet up and down 3 times, then incubate for 2 minutes at room temperature.
(For downstream processing on the KingFisher™mL instrument, you must mix by pipetting.)
4. Invert the tube of Lysis/Binding/Bead Mix several times to resuspend the beads, then add 720µL
of Lysis/Binding/Bead Mix to each sample.
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit
2
16 VetMAX™BVDV4ALLKitSupplemental Instructions
5. Immediately proceed to “Process samples on the instrument” on page19.
Note: If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, proceed to “Set
up the processing plates or tube strips (KingFisher™ Duo Prime and KingFisher™mL instruments)”
on page19.
Prepare samples for processing using the Simple Workflow
Prepare Bead/PKMix
We recommend that you prepare new Bead/PK Mix for each processing run. If necessary, you can store
Bead/PK Mix at 4°C for up to 1 week.
1. Vortex the MagMAX™ CORE Magnetic Beads thoroughly to ensure that the beads are fully
resuspended.
2. Combine the following components for the required number of samples, plus 10% overage
(recommended).
Component Volume per sample
MagMAX™ CORE Magnetic Beads 20µL
MagMAX™ CORE Proteinase K 10µL
Total Bead/PKMix 30µL
Prepare Lysis/Binding Solution
1. Combine the following components for the required number of samples plus 10% overage.
Component Volume per sample
MagMAX™ CORE Lysis Solution 350µL
MagMAX™ CORE Binding Solution 350 µL
Total Lysis/Binding Solution (–IPC) 700µL
5-IPC BVDV 4ALL[1] 5 µL
Total Lysis/Binding Solution (+IPC) 705µL
[1] Add 5-IPC BVDV 4ALL only to serum samples, and to BVDV positive control and NCS in extraction series that include at least
one serum sample.
2. Mix by inverting the tube or bottle at least 10times.
(Optional) Store Lysis/Binding Solution at room temperature for up to 24 hours.
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit 2
VetMAX™BVDV4ALLKitSupplemental Instructions 17
Prepare the sample
Prepare samples and controls as described.
Sample type Action
Individual samples Proceed with 200µL of sample.
Pooled samples Proceed with 200µL of pooled samples.[1]
BVDV positive control Proceed with 50µL of 4c-EPC BVDV 4ALL.
NCS —
[1] For information on the preparation of samples before extraction, see “Pooled serum or whole blood analyses” on page11.
Combine the sample with Bead/PK Mix, then add Lysis/Binding Solution
1. Invert the tube of Bead/PK Mix several times to resuspend the beads, then add 30µL of the
Bead/PK Mix to the required wells in the plate or tube strip.
2. Transfer each prepared sample to a well with Bead/PKMix.
3. Mix the sample with Bead/PK Mix for 2 minutes at room temperature according to your mixing
method.
•Using a plate shaker: shake vigorously for 2 minutes (see “(Optional) Determine the maximum
plate shaker setting” on page14).
•By pipetting: pipet up and down 3 times, then incubate for 2 minutes at room temperature.
(For downstream processing on the KingFisher™mL instrument, you must mix by pipetting.)
4. Add 700 µL of Lysis/Binding Solution to each sample-containing well or tube strip.
5. Immediately proceed to “Process samples on the instrument” on page19.
Note: If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, proceed to “Set
up the processing plates or tube strips (KingFisher™ Duo Prime and KingFisher™mL instruments)”
on page19.
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit
2
18 VetMAX™BVDV4ALLKitSupplemental Instructions
Set up the processing plates or tube strips (KingFisher™ Duo Prime and
KingFisher™mL instruments)
Note: When performing this procedure for processing on the KingFisher™mL instrument, mix samples
by pipetting up and down. Do not use a plate shaker with the tube strips required by this instrument.
1. Add each reagent to the indicated positions, according to your instrument.
Load the Tip Comb and all of the plates or tube strips at the same time. The instrument does not
prompt you to load items individually.
Table9Plate setup: KingFisher™ Duo Prime instrument
Row ID Row in the plate Plate type Reagent Volume per well
Sample A Deep Well Sample lysate/bead mix Varies by sample
Wash 1 B MagMAX™ CORE Wash Solution
1
500µL
Wash 2 C MagMAX™ CORE Wash Solution
2
500µL
Elution[1] Separate tube
strip[2]
Elution strip MagMAX™ CORE Elution Buer 90µL
Tip Comb H Deep Well Place a tip comb in the plate.
[1] Ensure that the elution strip is placed in the correct direction in the elution block.
[2] Placed on the heating element.
Table10Tube strip setup: KingFisher™mL instrument
Position ID Tube strip
position Tube Reagent Volume per well
Sample 1 Standard Sample lysate/bead mix Varies by sample
Wash 1 2 MagMAX™ CORE Wash Solution
1
500µL
Wash 2 3 MagMAX™ CORE Wash Solution
2
500µL
Elution 4 MagMAX™ CORE Elution Buer 90µL
Tip Comb N/A N/A Slide the tip comb into the tip comb holder.
2. Immediately proceed to “Process samples on the instrument” on page19.
Process samples on the instrument
1. Select the appropriate script on the instrument (see “Download the script” on page14).
2. Start the run, then load the prepared plates or tube strips in their positions when prompted by the
instrument.
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™ CORE Nucleic Acid Purification Kit 2
VetMAX™BVDV4ALLKitSupplemental Instructions 19
Store purified nucleic acid on ice for immediate use, at −20°C for up to 1month, or at −80°C for
long‑term storage.
Extraction with the MagMAX™‑96 Viral RNA Isolation Kit
Notes
The extraction protocol on magnetic beads given here is used with the MagMAX™ Express-24 Magnetic
Particle Processor.
Before starting
•Preparation of the TL lysis buer. Reconstitute the lysis buer as described below:
Components For 1 sample For N samples[1]
Lysis/Binding Solution
Concentrate
70µL N×70µL
Carrier RNA[2] 1µL N×1 µL
Vortex briefly
Add 100% isopropanol 70µL N×70µL
Vortex
[1] It is recommended to allow for an additional reaction with respect to the total number of extractions to be carried out (samples
+ controls).
[2] The carrier RNA may have a viscous appearance once thawed, which makes pipetting more difficult. In this case, place the
tube at 37°C for 10 to 15 minutes, vortex vigorously and centrifuge.
Once reconstituted, the TL lysis buer is stable for 1 month at room temperature; do not store
it at 2°C to 8°C as the carrier RNA may precipitate. If the TL is kept at 2°C to 8°C by mistake,
incubate for 10 to 15 minutes at 37°C and completely homogenize before use.
•For extraction series that include only serum samples, or blood samples and serum samples:
prepare TL+IPC lysis solution by adding 5 µL of 5 - IPC BVDV 4ALL to 140 µL of previously
prepared TL lysis buer. Prepare this solution just before use including an additional 10%. Discard
after use.
•Reconstitution of Wash buer 1: add the required quantity of 100% isopropanol to the Wash
Solution 1 Concentrate bottle according to the manufacturer’s recommendations prior to initial use.
•Reconstitution of Wash buer 2: add the required quantity of 96–100% ethanol to the Wash
Solution 2 Concentrate bottle according to the manufacturer’s recommendations prior to initial use.
•Reconstitution of the Mix Beads solution: each reaction requires 10µL of RNA Binding Beads and
10µL of Lysis/Binding Enhancer. This solution should be reconstituted just before use and then
discarded. Completely homogenize by gentle agitation and place at 2°C to 8°C until use.
•Prepare and identify the number of microtubes and extraction plates according to the number of
samples to be extracted (including negative and positive controls).
Chapter2RNA extraction from EDTA blood, serum
Extraction with the MagMAX™‑96 Viral RNA Isolation Kit
2
20 VetMAX™BVDV4ALLKitSupplemental Instructions
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Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

Brand
Thermo Fisher Scientific
Model
VetMAX BVDV 4ALL Kit
Type
Operating instructions
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