Multiplex Real-Time PCR Detection of Klebsiella pneumoniae Carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM-1) genes
Multiplex Real-Time PCR Detection of KPC & NDM-1 genes is available for download. pdf icon[PDF – 2 pages]
This procedure provides instructions for Taqman-based real-time PCR detection of blaKPC and blaNDM-1 in a single reaction from gram-negative bacteria. The universal 16S rRNA gene is used as a control for DNA extraction and amplification for each reaction. If desired, either KPC or NDM-1 can be assayed 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.
Use of trade names is for identification only and does not constitute endorsement by the Centers for Disease Control and Prevention.
This assay was validated on the ABI 7500 Fast real-time PCR instrument (Applied Biosystems, Inc., Foster City, CA) using a collection of NDM-1-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). This assay demonstrated 100% sensitivity & specificity for detection of NDM-1 and KPC.
|Equipment||Reagents & Media||Supplies|
|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 = A or G
- Probes are light-sensitive and should be shielded 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.
Run DNA lysates from the following control strains with each run:
- KPC- and NDM-negative control – K. pneumoniae ATCC strain BAA-1706
- KPC-positive control – K. pneumoniae ATCC strain BAA-1705
- NDM-positive control – K. pneumoniae ATCC strain BAA-2146
- No Template control (NTC) – sterile reagent grade type I water
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, pH8.0
- 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 rcf for 3 min.
- Transfer lysate (approx. 400 µl) into new, appropriately labeled centrifuge tube
- Store lysate at −20°C to −30°C until needed
- Thaw all reagents on ice.
- Prepare primer-probe mix containing a final concentration of 20 uM each primer and 10 uM each probe listed above. Store on ice, covered to protect from light. (Remainder can be frozen 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:
2× QuantiFast Probe PCR Master Mix – 10 µl
Primer-probe mix – 5 µl*
Sterile reagent grade H20 – 3 µl
Total volume = 18 µl Master Mix (+ 2 µl of template = 20 µl reaction)*Final concentrations: 500nM each primer and 250nM each probe
- Pipet 18 µl of the Final Master Mix into each appropriate 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.
- Ensure that the instrument is set up to detect the following reporter signals:
16s Universal: CY5
NDM 1 (HEX): VIC
- Thermal cycling conditions are as follows:
1) Enzyme activation step: 95°C for 3 minutes
2) 40 PCR cycles of :
Melt: 95°C for 3 seconds
Anneal/ extend: 60°C for 30 seconds
When analyzing the results, it is important to only consider amplification between 10-30 cycles as positive. Amplification prior to 10 cycles means the template should be diluted before repeating. Amplification after 30 cycles can indicate trace contamination. The no template control (water) control should not yield a product (Ct >40) but may produce a trace 16S result in the 31-40 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. Positive and negative controls should yield results described below.
|16S (CY5) Result||KPC (FAM) Result||NDM (HEX) Result||Report|
|Ct 10-30||Ct 10-30||Undetected||Positive||Negative|
|Ct 10-30||Undetected||Ct 10-30||Negative||Positive|
|Ct 10-30||Ct 10-30||Ct 10-30||Positive||Positive|
|Ct 10-30||Ct <10 (in either)||Dilute template 1:100, repeat|
|Ct <10 or Ct >40||anything||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. 22.214.171.124−126.96.36.199.
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.
Disclaimer: Use of trade names is for identification only and does not constitute endorsement by the Centers for Disease Control and Prevention.