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Laboratory Testing for Klebsiella pneumoniae Carbapenemase (KPC) and New Delhi metallo-\u03b2-lactamase (NDM) in Gram-negative Bacteria

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Key points

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

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The Klebsiella pneumoniae carbapenemase (KPC) is an Ambler class A beta-lactamase (\u03b2-lactamase) that confers (gives) resistance to most \u03b2-lactam agents, including carbapenems. The New Delhi metallo-\u03b2-lactamase (NDM) is an Ambler class B metallo-\u03b2-lactamase that can break down all \u03b2-lactams except the drug aztreonam.

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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.

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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.

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Test methods

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Validation

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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).

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

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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.

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Materials needed

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

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OligonucleotideNucleotide sequence, 5\u2019\u20133\u2019
KPC-F PrimerGGC  CGC  CGT  GCA  ATA  C
KPC-R PrimerGCC  GCC  CAA  CTC  CTT  CA
KPC-Probe (FAM)FAM-TG   ATA  ACG  CCG  CCG  CCA  ATT  TGT-BHQ
NDM-F PrimerGAC  CGC  CCA  GAT  CCT  CAA
NDM-R PrimerCGC  GAC  CGG  CAG  GTT
NDM-Probe (HEX)HEX-TG  GAT  CAA  GCA  GGA  GAT-BHQ
16S rRNA-FTGG  AGC  ATG  TGG  TTT  AAT  TCG  A
16S rRNA-RTGC  GGG  ACT  TAA  CCC  AAC  A
16S rRNA-Probe (CY5)CY5-CA  CGA  GCT  GAC  GAC  AR*C  CAT  GCA-BHQ
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* R = Mixture of A and G at this position, not necessarily equimolar

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Laboratory guidelines

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Positive controls and expected outcomes

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ControlFrequency (every run, every extraction, etc.)Expected Value
KPC - Positive Control
K. pneumoniae ATCC BAA-1705		Every runPositive: Ct 10 \u2013 30; KPC (+); NDM (-)
NDM - Positive Control
K. pneumoniae ATCC BAA-2146		Every runPositive: Ct 10 \u2013 30; KPC (-); NDM (+)
Spiked NDM and KPC Positive Control
BAA-1705 and BAA-2146 combination (50:50)		Every runPositive: Ct 10 \u2013 30; KPC (+); NDM (+)
16S Extraction (internal) Positive control (EPC)Every sample extraction and run16S Positive for each specimen tested; Ct 10-30 (except NTC & ENC controls which would be Ct >30 or Undetected)
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Negative controls and expected outcomes

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

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

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  1. Resuspend a 1 \u00b5l loopful of bacterial growth in 25 \u00b5l of sterile reagent-grade water in a labeled 1.5 ml centrifuge tube.
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  2. Add 25 \u00b5l of 0.1 N NaOH to each sample and mix by inverting.
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  3. Heat at 95\u201399\u00b0C for 10 minutes.
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  4. Cool 3-5 minutes on ice, then neutralize by adding 18 \u00b5l of 0.5 M Tris-HCl, pH 8.0.
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  5. Add 400 \u00b5l of cold, sterile reagent grade water to each tube.
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  6. Mix tube by inversion and flicking with your finger. Centrifuge at 16.1 relative centrifugal force (rcf) for 3 min.
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  7. Transfer lysate (approx. 400 \u00b5l) into new, appropriately labeled centrifuge tube.
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  8. Store lysate at 2\u00b0C to 8\u00b0C if testing on the same day, otherwise it should be kept at \u221215\u00b0C to \u221225\u00b0C until needed.
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Performing the KPC NDM real-time PCR assay

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  1. Thaw all reagents on ice.
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  2. Prepare primer-probe mix containing a final concentration of 20 \u00b5M of each primer and 10 \u00b5M of each probe listed above. Store on ice, covered to protect from light. (Can freeze remainder for subsequent use.)
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  3. Prepare Master Mix below to yield one 20 \u00b5l 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: \n
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    1. KiCqStart Probe qPCR ReadyMix, ROX \u2013 10 \u00b5l
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    2. Primer-probe mix \u2013 5 \u00b5l*
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    3. Sterile molecular grade (nuclease-free) water \u2013 3 \u00b5l
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    4. Total volume = 18 \u00b5l Master Mix (+ 2 \u00b5l of template = 20 \u00b5l reaction)
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  4. Pipet 18 \u00b5l of the Final Master Mix into each well in the 96-well plate.
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  5. Add 2 \u00b5l of template to respective wells. Ensure that the no template control is added last. Pipet up and down to mix.
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  6. Cover 96-well plate securely with optical adhesive film.
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  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).
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*Final concentrations: 500nM each primer and 250nM each probe

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Real-time PCR assay setup and run

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

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ProbeReporterDetector
16S UniversalCY5CY5
KPCFAMFAM
NDMHEXVIC
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Note: On the ABI 7500 instrument the reporter signal from the HEX-labeled NDM Probe is detected using the VIC channel.

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Thermal cycling conditions:

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Enzyme Activation Step95\u00b0C for 3 minutes
PCR Cycles
Melt
Anneal/Extend		35
95\u00b0C for 3 seconds
60\u00b0C for 30 seconds
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Special precautions

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

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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.

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If PCR controls pass, interpret PCR samples as follows:

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16S (CY5) ResultKPC (FAM) ResultNDM (HEX) ResultReport
Ct 10-30Ct 10-30UndetectedKPC-positive and NDM-negative
Ct 10-30UndetectedCt 10-30KPC-negative and NDM-positive
Ct 10-30Ct 10-30Ct 10-30KPC-positive and NDM-positive
Ct 10-30UndetectedUndetectedKPC-negative and NDM-negative
Ct 10-30Ct <10 (in either)Ct <10 (in either)Dilute template 1:100, repeat
Ct <10 or Ct >30Any valueAny valueConsult supervisor
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Resources

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\n Sources \n Print \n Share \n
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\n Facebook \n LinkedIn \n Twitter \n Syndicate \n
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\n National Center for Emerging and Zoonotic Infectious Diseases (NCEZID) \n
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  • Applied Biosystems 7500 Fast Real-Time PCR System Guide
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  • 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\u221212.5.4.10.
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  • Yong, D., M.A. Toleman, C.G. Giske et al. 2009. Characterization of a new metallo-\u03b2-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.
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Syndicated Content Details:
Source URL: http://www.cdc.gov/HAI/settings/lab/kpc-ndm1-lab-protocol.html
Source Agency: Centers for Disease Control and Prevention (CDC)
Captured Date: 2016-05-23 21:56:39.0
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