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
Equipment | Reagents & Media | Supplies |
---|---|---|
Real-time PCR system and analysis software |
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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) |
Negative 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 (-) |
Preparing sample lysates
Note: Select several colonies of pure overnight growth from a trypticase soy agar plate with 5% sheep's blood.
- Resuspend a 1 µl loopful of bacterial growth in 25 µl of sterile reagent-grade water in a labeled 1.5 ml centrifuge tube.
- Add 25 µl of 0.1 N NaOH to each sample and mix by inverting.
- Heat at 95–99°C for 10 minutes.
- Cool 3-5 minutes on ice, then neutralize by adding 18 µl of 0.5 M Tris-HCl, pH 8.0.
- Add 400 µl of cold, sterile reagent grade water to each tube.
- Mix tube by inversion and flicking with your finger. Centrifuge at 16.1 relative centrifugal force (rcf) for 3 min.
- Transfer lysate (approx. 400 µl) into new, appropriately labeled centrifuge tube.
- 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.
Performing the KPC NDM real-time PCR assay
- Thaw all reagents on ice.
- 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.)
- 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:
- KiCqStart Probe qPCR ReadyMix, ROX – 10 µl
- Primer-probe mix – 5 µl*
- Sterile molecular grade (nuclease-free) water – 3 µl
- Total volume = 18 µl Master Mix (+ 2 µl of template = 20 µl reaction)
- KiCqStart Probe qPCR ReadyMix, ROX – 10 µl
- Pipet 18 µl of the Final Master Mix into each well in the 96-well plate.
- Add 2 µl of template to respective wells. Ensure that the no template control is added last. Pipet up and down to mix.
- Cover 96-well plate securely with optical adhesive film.
- 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
Real-time PCR assay setup and run
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 |
Special precautions
- 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:
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 | Any value | Consult supervisor |
- 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.