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ARM-D for β-Lactamase ID
™
INSTRUCTIONS FOR USE
INTENDED USE
The ARM-D for β-Lactamase ID kit is a qualitative PCR-based molecular test for the detection of family-specific
KPC, CTX-M ESBLs, MBL, and ampC gene targets. Positive identification of the gene by this test indicates
the presence of the detected β-Lactamase gene. The assay involves extraction of DNA from bacterial cells
and subsequent PCR amplification with gel-based detection. The kit can be used for active surveillance of
antimicrobial resistance patterns and be beneficial for infection control. The β-Lactamase ID kit generates data
in just hours, when used with the Philisa® Thermal Cycler, compared to 24-48 hours for traditional phenotypic
methods. This product has not been cleared by the U.S. Food and Drug Administration for In Vitro Diagnostic use.
The product is For Research Use Only. Not for use in diagnostic procedures.
INTRODUCTION
Bacterial resistance to antibiotics poses a global threat to public health and in recent years mortality rates
associated with infections resistant to antibiotics has increased with the potential to spread through the patient
population. Of these resistance mechanisms, β-Lactamases are enzymes that cleave β-Lactam rings rendering
the β-Lactam family of antibiotics ineffective for treatment of clinically-important Gram-negative bacterial
infections. Specifically, β-Lactamases confer resistance to penicillins, cephamycins, extended-spectrum
β-Lactams and, in some cases, carbapenems. β-Lactam resistant Gram-negative organisms, producing multiple
or plasmid-mediated β-lactamases, are difficult to identify phenotypically and necessitate more specific detection
methods to identify clinically important β-lactamases. Genetic identification of these resistance mechanisms
is critical for active surveillance and infection control. Because these antibiotics are often selected for the
management and prevention of infectious disease, the presence and characteristics of specific β-Lactamases
can play a critical role in selecting the appropriate antibiotic therapy.
DETECTION
Prepare a 2.5% agarose gel. Agarose gels can be pre- or post-stained with ethidium bromide or equivalent
nucleic acid stain (see assay recommendations for optimal procedure). Load 5 µl of each PCR product into the
desired wells. Run the gel at 8-10 V/cm for approximatley 45 minutes to achieve sufficient resolution of control
bands. Following electrophoresis, stain the gel with ethidium bromide (or equivalent stain for nucleic acids). For
an accurate sizing comparison of sample products, the control reaction products may be loaded in multiple gel
lanes and compared to a DNA ladder (see example gel for exact sizing of each amplicon and approximate band
migration pattern).
INTERPRETATION
AmpC, CTX-M ESBLs, MBL, and KPC amplicons from unknown sample targets can be identified by comparing
the product sizes to the control reactions (A). The following example illustrates the comparison of various PCR
products from DNA extracted from isolates (B):
SUMMARY AND PRINCIPLES
Nucleic acid tests can provide supplemental resistance information to conventional culture susceptibility testing.
The ARM-D for β-Lactamase ID kit allows for identification of nine β-Lactamase ID gene families: IMP-1, NDM,
OXA-48, CTX-M-14, CTX-M-15, CMY-2, DHA, VIM, and KPC. This test utilizes sequence-specific primer pairs for
the PCR amplification of each target group. Additionally, an endogenous internal control that targets a conserved
region common in Gram-negative bacteria is included to reduce false negatives due to PCR inhibition, DNA
degradation, or poor extraction. Included with the kit are two external DNA control vials, containing synthetic DNA
templates, for multiplex PCR amplification of all nine targets between two reaction tubes.
After extraction of DNA from Gram-negative bacteria, rapid (22-minute) PCR amplification is performed on the Streck
Philisa Thermal Cycler using the ARM-D 2X Supermix. Agarose gel detection is used to separate PCR products and
compare the molecular weights against the external DNA control bands and a known molecular weight DNA ladder.
CONTENTS
The kit vials contain synthetic DNA oligonucleotide primers and DNA controls for the specified gene targets
suspended in TE buffer, pH 8.0. The kit contents are sufficient for 100 reactions total and 14 reactions of each
control DNA mix.
10X Primer Mix:
275 µL
Control DNA Mix #1:
14 µL
Control DNA Mix #2:
14 µL
PRECAUTIONS
1. For Research Use Only. Not for use in diagnostic procedures.
2. Use established precautions with potentially biohazardous specimens according to your laboratory guidelines.
3. Always use DNase/RNase-free plasticware/reagents and aerosol-barrier pipet tips.
4. SDS can be obtained at www.streck.com, by calling 800-843-0912, or by calling your local supplier.
STORAGE AND STABILITY
1. When stored at -20 °C, unused kit contents are stable through the expiration date.
2. Minimize the number of freeze-thaw cycles where possible. Aliquots of the reagents for long-term storage may
be prepared.
3. When using reagents for consecutive days, store at 4 °C. Store at -20 °C for extended storage periods.
SAMPLE EXTRACTION
The β-Lactamase ID kit was validated against DNA extracted from known clinical bacterial isolates using
QIAGEN® DNeasy® Blood and Tissue Kit (catalog #: 69504). Alternative nucleic acid extraction techniques/kits
should also give DNA of sufficient yield and quality. The 30-cycle PCR assay has not been tested for use with
clinical samples in which isolates are present in low numbers (e.g., direct, uncultured samples) or crude DNA
preparations (e.g., boil preps).
PCR PREPARATION
Thaw reagents and vortex briefly to mix contents. A brief centrifugation step may be needed to bring all volume to
the bottom of the tubes. Prepare a master mix (without template DNA) according to the table below based upon
the number of samples to be processed (plus one extra reaction). Include at least one of each control reaction
and a no-template-control (NTC) sample for each prepared master mix.
Component
25 µL Reaction
Final Concentration
Nuclease-Free Water
9 µL
Variable
ARM-D 2X Supermix
12.5 µL
1X
10X Primer Mix
2.5 µL
1X
1 µL
Variable
Template or Control DNA
LIMITATIONS
1. Due to competition for amplification reagents, the internal control may not be amplified in the presence of
large amounts of resistance gene target amplification. If a resistance gene target is amplified, the absence of
an internal control band should not be considered an invalid result.
2. The internal control primers have been designed to amplify a highly conserved gene target present in many
Gram-negative bacteria. However, the internal control may not successfully amplify from certain Gramnegative species or strains. Therefore, one should consider this in interpreting the absence of the internal
control product from a specific sample.
3. PCR product for the internal control may be detected in Control 2. Vectors for use in control tubes were
propagated in E. coli and may have residual amounts of carry-over E. Coli DNA present. This at times will result
in a weak gel band. This was not determined to affect detection of the gene of interest in the test isolates.
4. The gene family targets have been tested against a considerable number of isolates. The PCR primers will
only amplify the specified target families, and will not detect other β-Lactamases.
5. Using the β-Lactamase ID kit with other thermal cyclers or enzymes is possible, but optimization may be
required. Contact Streck Technical Services for assistance at 800-843-0912 or [email protected].
ASSAY RECOMMENDATIONS
1. UltraPure Agarose 1000 (Life Technologies, Ref #: 16550-100) was used for preparation of 2.5% agarose gels
used in optimization and validation studies.
2. 1X TAE Buffer was used for electrophoresis.
3. Agarose gels were stained with ethidium bromide post-electrophoresis. The ethidium bromide post stain was
made using 1 µl of a 10 mg/ml ethidium bromide stock per 1 ml of sterile water. Agarose gels were stained
for 30 seconds and de-stained in distilled water for 30 minutes.
ORDERING INFORMATION
Please call our Customer Service Department toll free 800-843-0912 for assistance. Additional information can
be found online at www.streck.com.
GLOSSARY OF HARMONIZED SYMBOLS
EC REP
LOT
REF
Authorized
Representative in
the European
Community
Batch
Code
Biological Risk
Catalog
Number
Manufacturer
Consult
Instructions
For Use
Temperature
Limitation
Use By
IVD
In Vitro Diagnostic
Medical Device
Glossary of symbols may contain symbols not used in the labeling of this product.
Aliquot 24 µL of master mix into Philisa® PCR Tubes. Add 1 µl of control DNA or test sample to each respective PCR tube.
PCR PROTOCOL
Execute the following protocol on the Philisa Thermal Cycler:
Philisa PCR Protocol
Hot-start
98 °C for 30 sec
30 cycles of:
98 °C for 5 sec
60 °C for 10 sec
72 °C for 20 sec
Final extension
72 °C for 30 sec
Runtime: 22 minutes
The brand and product names of the instruments are trademarks of their respective holders.
Streck 7002 S. 109 Street Omaha, NE 68128 USA
350649
2015-02