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 – 2 pages]

Background

This procedure provides instructions for Taqman-based real-time PCR detection of bla_KPC and bla_NDM-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.

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-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.

Materials needed

Equipment	Reagents & Media	Supplies
Real-time PCR system and analysis software	2x QuantiFast Probe PCR Kit (Qiagen) Primers and probes (below) Sterile reagent grade type I H2O	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

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

Special Precautions

• 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.

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Control Strains

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

Preparing Sample Lysates

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, pH8.0
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 rcf for 3 min.
7. Transfer lysate (approx. 400 µl) into new, appropriately labeled centrifuge tube
8. Store lysate at −20°C to −30°C until needed

Performing the KPC and/or NDM real-time Screening Assay

1. Thaw all reagents on ice.
2. 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.)
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:
   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

4. Pipet 18 µl of the Final Master Mix into each appropriate 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.

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Real-time PCR setup and run

1. Ensure that the instrument is set up to detect the following reporter signals:
   16s Universal: CY5
   KPC: FAM
   NDM 1 (HEX): VIC
2. 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

Interpretation of Results

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
			KPC	NDM
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	Undetected	Undetected	Negative	Negative
Ct 10-30	Ct <10 (in either)		Dilute template 1:100, repeat
Ct <10 or Ct >40	anything		Consult supervisor

References

Applied Biosystems 7500 Fast Real-Time PCR System Guide

Clinical Microbiology Procedures Handbook, 3^rd Ed., 2010. Garcia, L., editor. Detection of the bla_KPC 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, bla_NDM-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.

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