Laboratory Testing for Klebsiella pneumoniae Carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) in Gram-negative Bacteria

Key points

  • This procedure provides instructions for Taqman-based real-time PCR detection of blaKPC and blaNDM in a single reaction from gram-negative bacteria.

Overview

The Klebsiella pneumoniae carbapenemase (KPC) is an Ambler class A beta-lactamase (β-lactamase) that confers (gives) resistance to most β-lactam agents, including carbapenems. The New Delhi metallo-β-lactamase (NDM) is an Ambler class B metallo-β-lactamase that can break down all β-lactams except the drug aztreonam.

This procedure provides instructions for Taqman-probe-based real-time polymerase chain reaction (PCR) detection of blaKPC and blaNDM genes in a single reaction with lysates made from gram-negative bacteria. Use the universal 16S rRNA gene as a control for DNA extraction and amplification for each reaction. If desired, you can assay (laboratory test) either blaKPC or blaNDM independently by excluding the other set of primers and probe.

Although KPC and NDM appear to be the most common carbapenemases in the United States, it is important to note that there are other less common carbapenemases, as well as other mechanisms of carbapenem resistance.

Test methods

Validation

This assay was validated on the ABI 7500 Fast real-time PCR instrument (Applied Biosystems, Inc., Foster City, CA) using a collection of NDM-positive and KPC-positive isolates, as well as isolates containing other resistance mechanisms (i.e., AmpC, CTX-M, OXA, SME, VIM, IMP, GIM, SIM, SPM).

Laboratorians can modify this protocol as needed to use with other real-time PCR instruments and amplification reagents/kits following appropriate validation studies.

Disclaimer: Use of trade names is for identification purposes and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.

Materials needed

Materials needed
Equipment Reagents & Media Supplies
Real-time PCR system and analysis software
  • KiCqStart Probe qPCR ReadyMix, ROX (Sigma)
  • Primers and probes (below)
  • Sterile molecular grade (nuclease-free) water
  • Micropipettes and aerosol-free pipette tips
  • Sterile 1.5 ml microcentrifuge tubes
  • 96-well reaction plate appropriate for PCR system
  • Optical adhesive film
  • Ice bucket and ice

Primers & Probes

Primers & Probes
Oligonucleotide Nucleotide sequence, 5’–3’
KPC-F Primer GGC  CGC  CGT  GCA  ATA  C
KPC-R Primer GCC  GCC  CAA  CTC  CTT  CA
KPC-Probe (FAM) FAM-TG   ATA  ACG  CCG  CCG  CCA  ATT  TGT-BHQ
NDM-F Primer GAC  CGC  CCA  GAT  CCT  CAA
NDM-R Primer CGC  GAC  CGG  CAG  GTT
NDM-Probe (HEX) HEX-TG  GAT  CAA  GCA  GGA  GAT-BHQ
16S rRNA-F TGG  AGC  ATG  TGG  TTT  AAT  TCG  A
16S rRNA-R TGC  GGG  ACT  TAA  CCC  AAC  A
16S rRNA-Probe (CY5) CY5-CA  CGA  GCT  GAC  GAC  AR*C  CAT  GCA-BHQ

* R = Mixture of A and G at this position, not necessarily equimolar

Laboratory guidelines

Positive controls and expected outcomes
Control Frequency (every run, every extraction, etc.) Expected Value
KPC - Positive Control
K. pneumoniae ATCC BAA-1705		 Every run Positive: Ct 10 – 30; KPC (+); NDM (-)
NDM - Positive Control
K. pneumoniae ATCC BAA-2146		 Every run Positive: Ct 10 – 30; KPC (-); NDM (+)
Spiked NDM and KPC Positive Control
BAA-1705 and BAA-2146 combination (50:50)		 Every run Positive: Ct 10 – 30; KPC (+); NDM (+)
16S Extraction (internal) Positive control (EPC) Every sample extraction and run 16S Positive for each specimen tested; Ct 10-30 (except NTC & ENC controls which would be Ct >30 or Undetected)

Positive controls and expected outcomes
Control Frequency (every run, every extraction, etc.) Expected Value
NDM Negative Control
K. pneumoniae ATCC BAA-1705		 Every run Negative: Ct >30 or Undetected; KPC (+); NDM (-)
KPC Negative Control
K. pneumoniae ATCC BAA-2146		 Every run Negative: Ct >30 or Undetected; KPC (-); NDM (+)
KPC and NDM Negative Control
K. pneumoniae ATCC BAA-1706		 Every run Negative: Ct >30 or Undetected; KPC (-); NDM (-)
No Template Control (NTC)
Sterile molecular grade (nuclease free) water		 Every run Negative: Ct >30 or Undetected; KPC (-); NDM (-); 16S (-)
Extraction negative control (ENC)
molecular grade water, 0.1N NaOH, and 0.5M Tris- HCl		 Every run Negative: Ct >30 or Undetected; KPC (-); NDM (-); 16S (-)

Note: Select several colonies of pure overnight growth from a trypticase soy agar plate with 5% sheep's blood.

  1. Resuspend a 1 µl loopful of bacterial growth in 25 µl of sterile reagent-grade water in a labeled 1.5 ml centrifuge tube.
  2. Add 25 µl of 0.1 N NaOH to each sample and mix by inverting.
  3. Heat at 95–99°C for 10 minutes.
  4. Cool 3-5 minutes on ice, then neutralize by adding 18 µl of 0.5 M Tris-HCl, pH 8.0.
  5. Add 400 µl of cold, sterile reagent grade water to each tube.
  6. Mix tube by inversion and flicking with your finger. Centrifuge at 16.1 relative centrifugal force (rcf) for 3 min.
  7. Transfer lysate (approx. 400 µl) into new, appropriately labeled centrifuge tube.
  8. Store lysate at 2°C to 8°C if testing on the same day, otherwise it should be kept at −15°C to −25°C until needed.

  1. Thaw all reagents on ice.
  2. Prepare primer-probe mix containing a final concentration of 20 µM of each primer and 10 µM of each probe listed above. Store on ice, covered to protect from light. (Can freeze remainder for subsequent use.)
  3. Prepare Master Mix below to yield one 20 µl reaction for each test sample and control, and one additional reaction (to account for pipetting loss). Mix by flicking the tube; do not vortex. Each reaction contains:
    1. KiCqStart Probe qPCR ReadyMix, ROX – 10 µl
    2. Primer-probe mix – 5 µl*
    3. Sterile molecular grade (nuclease-free) water – 3 µl
    4. Total volume = 18 µl Master Mix (+ 2 µl of template = 20 µl reaction)
  4. Pipet 18 µl of the Final Master Mix into each well in the 96-well plate.
  5. Add 2 µl of template to respective wells. Ensure that the no template control is added last. Pipet up and down to mix.
  6. Cover 96-well plate securely with optical adhesive film.
  7. Conduct a quick spin of the sealed 96-well plate for approximately 10-20 seconds to force the liquid into the bottom of the reaction wells (with no bubbles).

*Final concentrations: 500nM each primer and 250nM each probe

Ensure that the instrument is set up to detect the following reporter signals:

Ensure that the instrument is set up to detect the following reporter signals
Probe Reporter Detector
16S Universal CY5 CY5
KPC FAM FAM
NDM HEX VIC

Note: On the ABI 7500 instrument the reporter signal from the HEX-labeled NDM Probe is detected using the VIC channel.

Thermal cycling conditions
Enzyme Activation Step 95°C for 3 minutes
PCR Cycles
Melt
Anneal/Extend		 35
95°C for 3 seconds
60°C for 30 seconds

  • Probes are light-sensitive; shield from light with foil.
  • Use aerosol-free pipette tips at all stages of testing to prevent contamination.
  • Use powder-free gloves as powder can cause unwanted fluorescence in this assay.

Interpreting results

When analyzing the results, it is important to only consider amplification between 10-30 cycles as positive. Amplification prior to 10 cycles means you should dilute the template before repeating. Amplification after 30 cycles can indicate trace contamination. The no template control (water) control should not yield a product (Ct >30) but may produce a trace 16S result in the 31-35 cycle range; both are acceptable. Amplification of any target other than 16S, or amplification of the 16S target below 30 cycles, in the no template control well indicates cross-contamination, resulting in an invalid run.

If PCR controls pass, interpret PCR samples as follows:

Interpreting Results
16S (CY5) Result KPC (FAM) Result NDM (HEX) Result Report
Ct 10-30 Ct 10-30 Undetected KPC-positive and NDM-negative
Ct 10-30 Undetected Ct 10-30 KPC-negative and NDM-positive
Ct 10-30 Ct 10-30 Ct 10-30 KPC-positive and NDM-positive
Ct 10-30 Undetected Undetected KPC-negative and NDM-negative
Ct 10-30 Ct <10 (in either) Ct <10 (in either) Dilute template 1:100, repeat
Ct <10 or Ct >30 Any value Consult supervisor

Resources

Print
Content Source:
National Center for Emerging and Zoonotic Infectious Diseases (NCEZID)
  • Applied Biosystems 7500 Fast Real-Time PCR System Guide
  • Clinical Microbiology Procedures Handbook, 3rd Ed., 2010. Garcia, L., editor. Detection of the blaKPC Gene Encoding Klebsiella pneumoniae Carbapenemase by Real-Time PCR. 12.5.4.1−12.5.4.10.
  • Yong, D., M.A. Toleman, C.G. Giske et al. 2009. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in K. pneumoniae sequence type 14 from India. Antimicrob. Agents Chemother. 43:5046-54.