Hygiena KIT230040 Brucella Detection LyoKit Instructions
- May 15, 2024
- Hygiena
Table of Contents
Hygiena KIT230040 Brucella Detection LyoKit
Product No. KIT230040
PCR system for 96 reactions for a maximum of 94 samples. Store the PCR kit at
-15 to -25 °C.
What This Product Does
Number of Tests
The detection system is designed for 96 reactions with a final reaction volume
of 25 μL each. Up to 94 samples (single sample preparation) plus positive and
negative control reactions can be analyzed per run.
Storage and Stability
- Store the kit at -15 to -25 °C through the expiration date printed on the label.
- Once the kit is opened, store the components as described in the following Kit Contents table:
Kit Contents
Vial/Cap Color| Label| Contents / Function /
Storage
---|---|---
1 yellow cap| foodproof Brucella
Master Mix
| • 3 x 600 µL
• Ready-to-use primer and Hydrolysis Probe mix specific for Brucella DNA and the Brucella -specific Internal Control (IC).
• For amplification and detection of Brucella -specific sequences.
• Store at -15 to -25°C.
• Avoid repeated freezing and thawing! Protect from light!
2 red cap| foodproof Brucella
Enzyme Solution
| • 3 x 32 µL
• Contains Taq DNA Polymerase and Uracil-DNA Glycosylase (heat labile) for prevention of carry-over contamination.
• Store at -15 to -25°C.
3 white cap| foodproof Brucella
Internal Control
| • 3 x 32 µL
• Contains a stabilized solution of plasmid DNA.
• For use as an internal amplification control.
• Store at -15 to -25°C.
• After first thawing store at 2 to 8 °C for up to one month.
4 purple cap| foodproof Brucella
Control Template
| • 1 x 50 µL
• Contains a stabilized solution of plasmid DNA.
• For use as a PCR run positive control.
• Store at -15 to -25°C.
• After first thawing store at 2 to 8 °C for up to one month.
5 colorless cap| H2O, PCR-grade| • 1 x 1 mL
• Nuclease-free, PCR-grade H2O.
• For use as a PCR run negative control.
• Store at -15 to -25°C.
Additional Equipment and Reagents Required
- Real-time PCR instruments with a FAM, VIC/HEX, ROX/Texas Red and Cy5 detection channel
- Real-time PCR compatible tubes, strips or plates with optical cap or foil applicable for the PCR cycler in use
- Nuclease-free, aerosol-resistant pipette tips
- Pipettes
- Sterile reaction tubes for preparing PCR mixes and dilutions
Applicability Statement
The foodproof Brucella Detection Kit is intended for the rapid detection of
Brucella spp., including the identification of Brucella abortus and Brucella
melitensis. The detection kit must not be used in diagnostic procedures. The
kit described in this Instruction Manual has been developed for real-time PCR
instruments with FAM, VIC/HEX, ROX/Texas Red and Cy5 detection channels. The
performance of the kit was tested with the following real-time PCR
instruments: Mx3000p® QPCR System (Stratagene), Mx3005 p® QPCR System
(Stratagene), LightCycler® 480 (Roche Diagnostics), Rotor-Gene® 6000 (Corbett
Life Science) and iQ5™ (Bio-Rad Laboratories).
Note: A Color Compensation (Color Compensation Set 3; Product No.
KIT230005) is necessary and will be supplied by Hygiena Diagnostics for users
of the LC 480 Systems I and II. Please contact Hygiena Diagnostics for further
information.
How to Use this Product
Before You Begin
Precautions
Detection of Brucella DNA using the foodproof Brucella Detection Kit requires
DNA amplification by PCR. The detection kit provides all the reagents required
for the PCR. In order to achieve reliable results, the entire assay procedure
must be performed under nuclease-free conditions. Follow the instructions
below to avoid nuclease-, carry-over-, or cross-contamination:
- Prepare appropriate aliquots of the solutions and keep them separate from other reagents in the laboratory.
- Use nuclease-free labware (e.g., pipettes, pipette tips, reaction vials).
- Wear gloves when performing the assay.
- To avoid cross-contamination of samples and reagents, use fresh aerosol-preventive pipette tips.
- To avoid carry-over contamination, transfer the required solutions for one experiment into a fresh tube, rather than directly pipetting from stock solutions.
- Physically separate the workplaces for DNA preparation, PCR setup, and PCR to minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps. Keep the foodproof Brucella Master Mix (vial 1, yellow cap) away from light.
Waste Disposal
Place any waste and biohazard material potentially contaminated with
pathogenic bacteria in an appropriate plastic Contaminated Waste bag and label
as follows: CONTAMINATED Waste, Room number, date and initials. The bag should
be autoclaved and then disposed of according to local regulations.
Sample Material
Use any sample material suitable for PCR in terms of purity, concentration,
and absence of inhibitors. For preparation of genomic DNA from raw material or
from food enrichments, refer to the corresponding product package inserts of a
suitable sample preparation kit.
Positive Control
Always run a positive control with the samples. To prepare a positive control,
replace the template DNA with the provided control DNA [foodproof Brucella
Control Template (vial 4, purple cap)] or with a positive sample preparation
control.
Negative Control
Always run a negative control with the samples. To prepare a negative control,
replace the template DNA with PCR-grade H2O (vial 5, colorless cap). Include a
negative control during sample preparation to monitor reaction purity and
cross-contamination. This extraction control can be used as an additional
negative control reaction.
For some real-time PCR instruments, the type of the probe quencher as well as the usage of a passive reference dye has to be specified. The food proof Brucella Detection Kit contains probes with a non-fluorescent (“dark”) quencher and no passive reference dye.
Note for users of the Agilent Mx3005P instrument:
Click ‘Instrument Filter Set Gain Settings’ to open the Filter Set Gain
Settings dialog box in which the gain settings may be viewed and modified. For
FAM, the Filter Set Gain Setting has to be modified to ‘x1’.
Preparation of the PCR Mix
Proceed as described below to prepare a 25 μL standard reaction. Always wear
gloves when handling the PCR vessels.
- Thaw the solutions. For maximal recovery of contents, briefly spin vials in a microcentrifuge before opening. Mix carefully but thoroughly by pipetting up and down.
- In a reaction tube (0.5 – 2.0 mL depending on the number of reactions), prepare the PCR Mix by adding the following components in the order mentioned below:
Note : The volumes indicated below are based on a single 25 μL standard reaction. Prepare the PCR mix by multiplying the amount in the “Volume” column by the number of reactions to be cycled plus one or two additional reactions to cover pipetting losses. Component| Volume
---|---
foodproof Brucella Master Mix (vial 1, yellow cap)| 18.0 µL
foodproof Brucella Enzyme solution (vial 2, red cap)| 1.0 µL
foodproof Brucella Internal Control (vial 3, white cap)| 1.0 µL
Total volume| 20.0 µL - Mix carefully but thoroughly by pipetting up and down. Do not vortex.
- Pipet 20 μL PCR mix into each PCR vessel.
- For the samples of interest, add 5 μL sample DNA.
- For the negative control, add 5 μL H2O, PCR-grade (vial 5, colorless cap).
- For the positive control, add 5 μL foodproof Brucella Control Template (vial 4, purple cap).
- Seal the PCR vessels accurately with optical caps or foil.
- Briefly spin the PCR vessels in a suitable centrifuge.
- Cycle the samples as described above
Data Interpretation
The amplification of DNA of Brucella abortus is analyzed in the fluorescence
channel suitable for FAM-labeled probes detection and of Brucella melitensis
in the detection channel for VIC/HEX. The amplification of DNA of any member
of the genus Brucella is analyzed in the fluorescence channel suitable for
ROX/Texas Red-labeled probe detection. The amplification signal of the
Positive Control can be detected in all three channels. The specific
amplification of the Internal Control (IC) is analyzed in the fluorescence
channel suitable for Cy5. Compare the results from channel FAM (Brucella
abortus), VIC/HEX (Brucella melitensis), ROX/Texas Red (Brucella), and channel
Cy5 (Internal Control) for each sample and interpret as described in the
following table.
Channel FAM| Channel VIC/HEX| Channel ROX|
Channel Cy5 (IC)| Result Interpretation
---|---|---|---|---
Positive| negative| Positive| Positive or Negative| Positive for Brucella
and B. abortus
Negative| positve| Positive| Positive or Negative| Positive for Brucella and
B. melitensis
Positive| positive| Positive| Positive or Negative| Positive for Brucella,
B. abortus and B. melitensis
Negative| Negative| Positive| Positive or Negative| Positive for Brucella
(non B. abortus or B. melitensis )
Negative| Negative| Negative| positive| Negative for Brucella, B.
abortus or B. melitensis respectively
Negative| Negative| Negative| Negative| Invalid
Note : A prerequisite for the unambiguous discrimination of Brucella abortus (FAM), Brucella melitensis (VIC/HEX), Brucella (ROX/TEXAS Red) and Internal Control DNA in this multi-color experiment is a suitable calibration of the PCR instrument for channels FAM, VIC/HEX, ROX/Texas Red and Cy5. Please refer to the operation manual for your real-time PCR cycler for further information.
Troubleshooting
| Observation | Possible Reason | Recommendation |
|---|---|---|
| No signal increase is observed, even with positive controls. | Incorrect | |
| detection channel has been chosen. | · Set Channel settings to FAM, VIC/HEX, |
ROX/Texas Red or Cy5.
Pipetting errors or omitted reagents.| • Check for correct pipetting scheme
and reaction setup. Repeat the PCR run.
• Always run a positive control along with your samples.
No data acquisition programmed.| · Check the cycle programs.
No signal increase in channel Cy5.| Inhibitory effects of the sample material
(e.g., caused by insufficient purification).| • Dilute samples or pipet a
lower amount of sample DNA (e.g., 2.5 µL instead of 5 µL, substitute with
H2O, PCR-Grade).
Fluorescence intensity is too low.| Inappropriate storage of kit components.| • Store the foodproof Brucella Master Mix (vial 1, yellow cap) at -15 to -25 °C, protected from light.
• Avoid repeated freezing and thawing.
foodproof Brucella Master Mix
(vial 1, yellow cap) is not homogeneously mixed.
| • Mix the foodproof Brucella Master Mix (vial 1, yellow cap) thoroughly
before pipetting.
Low initial amount of target DNA.| • Increase the amount of sample DNA.
Depending on the chosen DNA isolation method, inhibitory effects may occur.
Negative control samples are positive.| Carry-over contamination.| • Exchange
all critical solutions.
• Repeat the complete experiment with fresh aliquots of all reagents.
• Always handle samples, kit components and consumables in accordance with commonly accepted practices to prevent carry-over contamination.
• Add positive controls after sample and negative control reaction vessels have been sealed.
Fluorescence intensity varies.| Insufficient centrifugation of the PCR vessels.
Prepared PCR mix is still in the upper part of the vessel.
| • Always centrifuge reaction vessels.
Outer surface of the vessel or seal is dirty (e.g., by direct skin contact).|
• Always wear gloves when handling the vessel and seal.
Additional Information on this Product
How this Product Works
The foodproof Brucella Detection Kit provides primers and Hydrolysis Probes
(for sequence-specific detection), convenient premixed reagents, and a control
template for reliable interpretations of results. To ensure maximum
reliability of the detection system and to prevent misinterpretation of
negative results due to inhibition of the amplification, an Internal Control
(IC) is supplied (vial 3, white cap). The IC has to be added to each reaction.
A Hydrolysis Probe was designed to bind specifically to the IC, allowing
detection in the Cy5 channel, whereas the Brucella DNA is detected in the FAM
(B. abortus), VIC/HEX (B. melitensis) and ROX/Texas Red (genus Brucella)
channel. In case of a negative result due to inhibition of amplification by
the sample DNA of interest, the amplification of the IC is suppressed as well.
A negative result for the sample DNA of interest and amplification of the IC
clearly indicates the absence of Brucella DNA in the sample. The foodproof
Brucella Detection Kit minimizes contamination risk and contains all reagents
needed for detection of Brucella DNA. Primers and probes provide specific
detection of Brucella DNA in food samples. The kit described in this
Instruction Manual has been developed for real-time PCR instruments with a
FAM, VIC/HEX, ROX/Texas Red and Cy5 detection channel.
Test Principle
- Using the supplied sequence-specific primers in a polymerase chain reaction (PCR), the PCR instrument and its associated reagents amplify and simultaneously detect fragments of Brucella genomic DNA.
- The PCR instrument detects these amplified fragments in real time through fluorescence generated by cleavage of the hybridized probe due to the 5´ nuclease activity of the Taq DNA polymerase. The probe is labeled at the 5´-end with a reporter fluorophore and at the 3´-end with a quencher.
- During the annealing/elongation phase of each PCR cycle, the probe hybridizes to an internal sequence of the amplicon downstream from one of the primer sites and is cleaved by the 5´ nuclease activity of the Taq DNA polymerase. This cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal.
- The real-time PCR instrument measures the emitted fluorescence of the reporter dye.
Prevention of Carry-Over Contamination
The heat-labile Uracil-DNA Glycosylase (UNG) is suitable for preventing carry-
over contamination between PCRs. This technique relies on the incorporation of
deoxyuridine triphosphate (dUTP) during all amplification reactions and the
pretreatment of all successive PCR mixtures with the heat-labile UNG. The UNG
cleaves DNA at any site where a deoxyuridine residue has been incorporated.
The resulting abasic sites are hydrolyzed due to the high temperatures during
the initial denaturation step and can no longer serve as PCR templates. The
heat-labile UNG is inactivated during the initial denaturation step. Native
DNA (e.g., the isolated Brucella genomic DNA) does not contain uracil and is
therefore not degraded by this procedure. Since dTTP is replaced with dUTP and
UNG is included in the foodproof Brucella Detection Kit, decontamination can
be achieved with the provided reagents.
Background Information
The genus Brucella, member of the Brucellaceae family (class:
Alphaproteobacteria), comprises the six species B. abortus, B. melitensis, B.
suis, B. ovis, B. canis, and B. neotomae. Bacteria of the genus Brucella are
gramnegative, facultative intracellular, non-motile, non-spore forming
pathogens that infect a wide range of animal hosts and the species B. abortus,
B. melitensis, B. suis, and B. canis may also infect humans. Brucella sp. are
the causative organisms of the disease brucellosis in humans, also called
undulant fever, or Malta fever. Brucellosis is a highly contagious zoonosis
(infectious disease transmitted from animals to humans) caused by eating raw,
minced meat or contaminated or untreated milk (and its derivates) or through
direct contact with infected animals, which may include dogs, pigs, camels and
ruminants, primarily sheep, goats, cattle, bison. Common microbiological and
serological methods for the detection and identification of Brucella spp. are
very time-consuming and very hazardous for the laboratory staff [1, 2].
Therefore, several PCR ELISA and real-time PCR assays were developed to
fulfill the need for rapid, sensitive and specific systems for the detection
of Brucella spp. [3].
Product Specifications
Specificity:
The foodproof Brucella Master Mix is sequence-specific for highly conserved
genes found in all Brucella sp., in Brucella abortus, or Brucella melitensis.
Inclusivity has been tested with more than 50 strains of B. abortus, B.
melitensis, B. suis, B. canis, B. ovis and B. neotomae, where all of them
could be detected (100% inclusivity). Exclusivity was determined using 25
species of closely related organisms or organisms occurring in the same
habitat.
References
- Al Dahouk S., et al. (2003) Laboratory-based diagnostics of brucellosis – a review of the literature. Part I: techniques for direct detection and identification of Brucella spp. Clin Lab 49, 487-505.
- Al Dahouk S., et al. (2003) Laboratory-based diagnostics of brucellosis – a review of the literature. Part II: serological tests for brucellosis. Clin Lab 49, 577-589.
- Al Dahouk S., et al. (2004) The detection of Brucella spp. Using PCR-ELISA and real-time PCR assays. Clin Lab 50, 387-394.
Quality Control
The foodproof Brucella Detection Kit is function tested using the LightCycler
480 System.
Supplementary Information
Ordering Information
Hygiena Diagnostics offers a broad range of reagents and services. For a
complete overview and for more information, please visit our website at
www.hygiena.com.
License Notice
The purchase price of this product includes limited, nontransferable rights
under US Patent No. 7,687,247 owned by Life Technologies Corporation to use
only this amount of the product to practice the claims in said patent solely
for activities of the purchaser for bioburden testing, environmental testing,
food testing, or testing for genetically modified organisms (GMO) in
accordance with the instructions for use accompanying this product. No other
rights are conveyed, including no right to use this product for in vitro
diagnostic, therapeutic, or prophylactic purposes. Further information on
purchasing licenses under the above patent may be obtained by contacting the
Licensing Department, Life Technologies Corporation, 5791 Van Allen Way,
Carlsbad, CA 92008.
Email:outlicensing@lifetech.com
Trademarks
foodproof® is a registered trademark of Hygiena Diagnostics GmbH. Other brand
or product names are trademarks of their respective holders.
Contact and Support
If you have questions or experience problems with this or any other product of Hygiena Diagnostics GmbH, please contact our Technical Support staff (www.hygiena.com/support). Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing our product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to us and the worldwide research community.
Reference Number
The reference number and original Hygiena Diagnostics GmbH article number: R
302 04
Change Index
-
Version 1
First version of the package insert. -
Version 2
New product name extension: 5’ Nuclease. -
Version 3, July 2010
Page 7: Note for users of the Agilent Mx3005p instrument added. -
Version 4, March 2017
License Notice changed.
Revision A, January 2024
Rebranding and new layout.
R 302 04 20 -> INS-KIT230040-RevA
Hygiena®
Camarillo, CA 93012
USA
diagnostics.support@hygiena.com
Manufactured by
Hygiena Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
www.hygiena.com
Documents / Resources
|
Hygiena KIT230040 Brucella Detection
LyoKit
[pdf] Instructions
KIT230040 Brucella Detection LyoKit, KIT230040, Brucella Detection LyoKit,
Detection LyoKit
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