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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:

info@biolegio.com

For information regarding to the protocol:

schubert@med.uni-muenchen.de

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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.