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Carbapenemases-2 assay
For the detection of NDM, IMP 14-18 group(blaIMP-14,-18), VIM(blaVIM-1bis -
34) and GIM genes using the BD MAXTM system.
Instructions for use
(Version 1.0 – March 2017)
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Contents
Introduction
Contact information
1. Protocol
1.1. Materials required
1.2. Run settings
1.3. Sample preparation
1.4. Setting up the experiment
2. Result interpretation
3. Data of Test-Validation
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Introduction1
Carbapenemases are β-lactamases with versatile hydrolytic capacities. They have the ability to
hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria producing
these β-lactamases may cause serious infections in which the carbapenemase activity renders
many β-lactams ineffective. Carbapenemases are members of the molecular class A, B, and D β-
lactamases. Class A and D enzymes have a serine-based hydrolytic mechanism, while class B
enzymes are metallo-β-lactamases that contain zinc in the active site. The class A
carbapenemase group includes members of the IMI, GES, and KPC families. The class D
carbapenemases consist of OXA-type β-lactamases such as the OXA48 like carbapenemases,
The metallo-β-lactamases belong to the IMP, VIM, SPM, GIM, and SIM families and have been
detected primarily in Pseudomonas aeruginosa; however, there are increasing numbers of
reports worldwide of this group of β-lactamases in the Enterobacteriaceae.
1. Queenan, A. M. and K. Bush. 2007. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev. 20:440-
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This protocol describes the system settings and setup protocols for running the Carba-2 panel to
detect four pathogens using the BD MAXTM system (assay target genes):
1. NDM
2. IMP 14-18 group (blaIMP-14,-18)
3. VIM(blaVIM-1bis -34)
4. GIM
The qPCR has been exclusively validated for bacterial culture from agar plates, e.g. MacConkey
agar or Bloodagar.
Contact information
For information regarding ordering dried snap-in tubes for the Carba-2 assay:
For information regarding to the protocol:
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1. Protocol
This protocol describes the assay settings required for running the Carba-2 assay on the BD
MAXTM system. The Carba-2 snap-in assay contains primers and probes for the detection of
NDM, IMP 14-18 group, VIM and GIM.
Materials needed
BD MAXTM system
BD MAXTM ExK DNA-2 Extraction Kit (BD cat.no: 442820)
BD MMK Mastermix with SPC (BD cat.no: 442829)
BD MAXTM PCR Cartridges (BD cat.no: 437519)
Dried snap-ins Carba-2 (Biolegio cat no: BDT-14018)
Eppendorftubes 1,5 ml
Vortex Mixer
Micropipettes
Safeseal Filtertips
Disposable gloves
1.1 Run settings
The assay is performed on the BD MAXTM system with use of the “BD MMK with SPC” in
combination with the “ExK DNA-2 Kit” for the extraction.
Create a full process assay in the test editor named “Carba-2 assay”, use the corresponding
Extraction type “2” (as shown in the screenshot of “Basic Information”) and the following
parameters:
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Edit the test steps using the following settings:
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1.3. Sample preparation
Bacterial culture
Take 2-4 colonies cultivated on agar plate (e.g. MacConkey agar) and dispense them
in 500 µl A. dest Prepare a 1:100 Dilution
Transfer 200 µl of the 1:100 diluted sample into a BD MAXTM DNA Sample Buffer
Tube and close the tube with a blue septum cap. Ensure complete mixing by
vortexing the sample
Extraction will be done with the “BD MAXTM ExK DNA-2 Extraction Kit
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1.4. Setting up the experiment
a. Create a worklist on the BD MAX instrument using the Carba-2 assay (created in step
1.2) and label the lanes with the sample names.
b. Load the prepared Sample Buffer Tubes into their corresponding position in the BD
MAX racks.
c. Load the BD MAX racks with the corresponding Unitized Reagent Strip.
d. Snap in the BD Extraction tubes (position 1), MMK tubes (position 2) and Carba-2
tubes (position 3) into the Reagent Strip.
e. Load the racks and cartridges into the BD MAX and Start Run
2. Result interpretation
2.1 For a run to be valid
No BD MAX System failures.
Negative Control (optional) has a Cq value of -1 for all channels
Positive control (optional) has a Cq value for channel 475/520 , 530/565,
585/ 630, 630/665 and 680/715
2.2 Interpretation if run is valid
A Cq value of -1 indicates a negative result
A Cq value for either of the targets indicates a positive result for the
corresponding target.
The SPC (channel 680/715) should always give a Cq value, if there is no other target
positive. A negative value for the SPC together with negative Cq value in every
other channel indicates inhibition. Therefore this sample should be repeated.
All curves need to be visually checked for right interpretation.
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3. Validation
3.1 Targets
3.1.1. NDM: (GenBank Acc. No.: FN396876.1)
3.1.2. IMP 14-18 group: (GenBank Acc. No.: NG049177)
3.1.3. VIM: (GenBank Acc. No.: KX641710)
3.1.4. GIM: (GenBank Acc. No.: NG049144)
3.2. Sensitivity / Specificity / Analytical Sensitivity
The sensitivity and specificity was determined using 15 defined positive and 25
defined negative samples, which were unequivocally assessed positive and
negative by 2 independent other PCRs, respectively.
The analytical sensitivity was determined by log-step dilutions of bacterial strains
(CFU) carrying the respective DNA target sequences.
All PCRs have been used in our clinical microbiological diagnostic laboratory for
the last 4 years under accreditation. They having passed constantly external
inter-laboratory comparison programmes (twice a year).
Target analytical sensitivity (bacterial culture)
NDM 103 copies/ ml
IMP 14-18 group 103 copies/ ml
VIM 103 copies/ ml
GIM 103 copies/ ml
Disclaimer:
MvP-Institut is not responsible for the results on the Carba-2 assay on the BD MAX system.
Using the „open protocol“, the respective laboratory itself is responsible for the validation of
the assay.