For Veterinary Use Only. For In Vitro Use Only.
INSTRUCTIONS FOR USE
VetMAX™ Neospora caninum Detection Kit
TaqMan® real-time PCR detection of
Neospora caninum
Catalog Number NEOSP
Doc. Part No. 100032698 Pub. No. MAN0010879 Rev. B.0
Technology
Species
Samples
Test type
Real-time PCR (DNA)
– Duplex assay
– Exogenous IPC
Bovine
Small ruminants
(sheep, goat)
Tissues
(brain, heart muscle, abortive tissues) Individual
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.
Product description
The Applied Biosystems™ VetMAX™ Neospora caninum Detection Kit (Cat. No. NEOSP) enables detection of Neospora caninum in
ruminant tissues by real-time PCR amplification of the Neospora Nc5 gene.
The assay is a single-well real-time PCR in which Neospora and exogenous Internal Positive Control (IPC) targets are amplified and
detected using fluorescent TaqMan®probes. The kit includes:
•3 - Mix Neospora: master mix for optimized duplex real-time PCR amplification of Neospora and IPC targets. It contains primers, TaqMan®
probes, and PCR buffer and enzyme.
•4a -EPC Neospora: nucleic acid template for Nc5 target. It serves as an external positive control for the real-time PCR components, and it
is used to set the threshold cycle (Ct) for evaluating test results.
•5 - IPC: internal positive control that is added to each sample and control at the lysis step of the DNA extraction procedure. It serves as a
control for the DNA purification process, and it is used to monitor for the presence of PCR inhibitors.
Kit contents and storage conditions
Reagents for 50 real-time PCR reactions (25 µL) are supplied.
Component Volume
(50 reactions)
Storage
3 - Mix Neospora 2 × 500 µL −30°C to −10°C
4a - EPC Neospora 1 × 90 µL −30°C to −10°C
5 - IPC(1) 1 × 250 µL −30°C to −10°C
(1) Intended for use only with VetMAX™ Neospora caninum Detection Kit.
2 VetMAX™ Neospora caninum Detection Kit Instructions for Use
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other
major laboratory supplier.
Item Source
Instrument and equipment
Applied Biosystems™ 7500 Real-Time PCR System, or equivalent Contact your sales representative
Nuclease-free pipettes MLS
2 ice buckets or refrigerated racks:
•One for the PCR setup area where the PCR master mix is prepared
•One for the area where DNA samples and controls are prepared
MLS
Precision Plate Holder for 7500 Real-Time PCR System, as appropriate for your experiment
For plates A24820
For 0.2-mL tube strips
4367033
Optical reaction plates and covers, or optical PCR tubes and caps, as appropriate for your experiment
MicroAmp
™
Optical 96-Well Reaction Plate
4316813
MicroAmp
™
Optical Adhesive Film
4311971
MicroAmp™ Optical 8-Tube Strip, 0.2 mL 4316567
MicroAmp™ Optical 8-Cap Strips 4323032
Reagents
Nuclease-Free Water AM12450
1X TE buffer
MLS
Other consumables
Nuclease-free reagent tubes for preparing master mix MLS
Filtered pipette tips
MLS
Important procedural guidelines
•For each real-time PCR run, include the controls indicated in step 1.
•Thaw amplification reagents in one ice bucket or refrigerated rack and controls and samples in a separate ice bucket or refrigerated rack.
•Keep thawed reagents, controls, and samples cooled (2–8ºC).
•Thoroughly mix the contents of each tube by vortexing, then briefly centrifuge before use.
•Follow “Good laboratory practices for PCR” on page 4 to prevent false positives and contamination of test samples with PCR products.
Input DNA requirements
The assay is performed on DNA extracted from 20 mg of tissue. DNA extraction methods that are validated for use with the
VetMAX™ Neospora caninum Detection Kit are available from technical support upon request. In addition, prepare mock-purified
sample using Nuclease-Free Water as starting material and the same DNA isolation method used for test samples (extraction control).
IMPORTANT! Add 5 µL of 5 - IPC to the lysis solution used for DNA isolation for each sample and extraction control.
Perform real-time PCR
Set up the real-time PCR reactions
1. Dispense 20 µL of 3 - Mix Neospora to the appropriate number of PCR plate wells or strip tubes
2. Add sample or control according to the following table:
Sample type Component Volume per reaction
Test sample
Sample DNA
5.0 µL
Positive control 4a - EPC Neospora 5.0 µL
Extraction control Mock-purified sample 5.0 µL
No-template control
Nuclease-Free water
5.0 µL
3. Seal each reaction vessel.
VetMAX™ Neospora caninum Detection Kit Instructions for Use 3
Set up and run the real-time PCR instrument
1. Following the manufacturer’s instructions, set up the real-time PCR run using the following parameters.
•Reaction volume: 25 µL
•Passive reference: ROX™ dye (included in 3 - Mix Neospora).
Note: ROX™ dye must be set up if the thermal cycler is capable of detecting it. Thermal cyclers that do not detect ROX™ dye
may be used without affecting the accuracy of the reading.
•Select detectors and assign TaqMan® probe reporter dyes and quenchers for each well or tube used in the analysis:
Target Reporter Quencher
Neospora
FAM™ dye NFQ (Non-Fluorescent Quencher)
IPC
VIC
™
dye
TAMRA
™
dye
(1)
(1) TAMRA™ dye must be set up for real-time PCR analysis if the thermal cycler is capable of detecting it. Thermal cyclers that do not detect TAMRA™
dye may be used without affecting the accuracy of the reading.
•Thermal cycling parameters:
Stage Repetitions Temperature Time
1
1×
50ºC
2 minutes
2
1×
95ºC
10 minutes
3 45× 95ºC 15 seconds
60ºC
1 minute
2. Run the thermal cycler program and collect real-time amplification data during the elongation step (1 minute at 60ºC).
Data analysis
Refer to the recommendations of the thermal cycler manufacturer for raw data analysis.
1. Set thresholds separately for each real-time PCR target.
2. Interpret the results for each control and sample according to the obtained Ct values as indicated in the following sections.
Validation
Refer to the Ct QC values listed in the Certificate of Analysis of the batch used for the test. The run is validated if the following criteria
are met:
Control reaction
Neospora target
(FAM
™
dye)
IPC target
(VIC
™
dye)
Interpretation
Positive control Ct = Ct QC
Neospora
± 3Ct(1) Any value(2) PCR validated
Extraction control
(3)
C
t
> 45
C
t
= C
t QC
IPC ± 3C
t(4)
Extraction validated
No-template control Ct > 45 Ct > 45 PCR reagents validated
(1) Refer to the values listed in section “Quality control results - EPC” of the Certificate of Analysis.
(2) The IPC value of the positive control is not used for test validation.
(3) Use the extraction control prepared in the same extraction procedure as the samples.
(4) Refer to the value listed in section “Quality control results - IPC” of the Certificate of Analysis.
Interpretation of results
Neospora
target
(FAM
™
dye)
IPC target
(VIC
™
dye)
Interpretation
Ct < 45 Any value Neospora caninum detected
C
t
> 45
C
t
= C
t
of extraction control ±3C
t(1)
Neospora caninum
not detected
Ct > 45 Ct is outside this range: Ct of extraction control ±3 Ct(1) Not validated(2)
(1) The Ct value of the extraction control must first be validated as described in “Validation”.
(2) The result is invalid due to a non-compliant IPC result.
Handling of non-validated samples
1. Dilute the DNA of non-validated samples 1:10 in 1X TE buffer.
2. Perform real-time PCR analysis with 5 µL of this dilution.
3. If the diluted DNA is positive for Neospora or negative for Neospora with a compliant IPC result, the result is then validated.
4. If the diluted DNA is negative for Neospora with a non-compliant IPC result, the result is still not validated. In this case, repeat
the analysis on a new sample.
thermofisher.com/support | thermofisher.com/askaquestion
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24 July 2018
Good laboratory practices for PCR
When preparing samples for PCR amplification:
•Wear clean gloves and a clean lab coat (not previously worn while handling amplified products or used during sample preparation).
•Change gloves whenever you suspect that they are contaminated.
•Maintain separate areas and dedicated equipment and supplies for:
•Sample preparation and reaction setup.
•Amplification and analysis of products.
•Do not bring amplified products into the reaction setup area.
•Open and close all sample tubes carefully. Avoid splashing and spraying samples.
•Keep reactions and components capped as much as possible.
•Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.
•Clean lab benches and equipment periodically with 10% bleach solution or DNAZap™ Solutions (Cat. No. AM9890).
Documentation and support
Customer and technical support
Technical support: visit thermofisher.com/askaquestion
Visit thermofisher.com/support for the latest in services and
support, including:
•Worldwide contact telephone numbers
•Order and web support
•User guides, manuals, and protocols
•Certificates of Analysis
•Safety Data Sheets (SDSs; also known as MSDSs)
NOTE: For SDSs for reagents and chemicals from other
manufacturers, contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant
their products as set forth in the Life Technologies' General
Terms and Conditions of Sale found on Life Technologies'
website at www.thermofisher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please
contact Life Technologies at thermofisher.com/support.
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES 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 of Pub. No. MAN0010879 (English)
Revision
Date
Description
B.0
24 July 2018
Updated to the current document template, with associated updates to the warranty, trademarks, and logos.
A.0
7 April 2015
New document.
Limited Use Label License No. 460: PCR Veterinary Diagn ostics. Notice to Purchaser: For veterinary diagnostic services including the reporting of such services for a fee and
for research purposes only. Human diagnostic uses require a separate license from Roche.
©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan
is a registered trademark of Roche, used under permission and license.
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