Feb 08, 2022

Public workspaceIdentification of the different phylogroups from the Klebsiella pneumoniae species complex by real-time PCR

Microbiology Spectrum
DOI
dx.doi.org/10.17504/protocols.io.b4s5qwg6
  • 1Université de Bourgogne Franche-Comté - INRAe Dijon - France;
  • 2Institut Pasteur, Université de Paris, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France;
  • 3INRAE, UR OPAALE, F-35044, Rennes, France
  • Klebsiella Research and Surveillance
Elodie Barbier
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DOI: dx.doi.org/10.17504/protocols.io.b4s5qwg6
Protocol Citation: Elodie Barbier, Carla Rodrigues, Sylvain Brisse, Pascal Piveteau 2022. Identification of the different phylogroups from the Klebsiella pneumoniae species complex by real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.b4s5qwg6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 08, 2022
Last Modified: February 08, 2022
Protocol Integer ID: 57917
Keywords: Klebsiella ; phylogroups; PCR ; identification
Abstract
KpSC phylogroup ID

We developed and optimized a real-time PCR for the identification of the different phylogroups (Kp1 to Kp6) belonging to Klebsiella pneumoniae species complex (KpSC) by amplification of five specific gene targets.
Primers and Probes
Primers and Probes

The KpSC phylogroup ID : Primers, Probes and Amplicon length

ABCDEFG
PhG -Target Forward (5‘ – 3‘) Reverse (5‘ – 3‘) Probe Quencher Reporter Amplicon length (bp)
Kp1 - group_8970 CCTCAAACACGCCAATATGC TACCGCGACGAGTAAAGTGG GATCCATTGATTCCATTCGAACCGG BHQ-1 JOE 85
Kp2 - group_10724 TTGTTGATTGGCAGGCCTTC AAATGCTGTGACCACCGTTG CGCCTGGCGTCAGTGGCCCCGAGC BHQ-1 JOE 70
Kp3/Kp5 - tetR GCGGGCCGGCTTTTC CGCATCCCAGGGTATATTCG AGGCCGAAGCCGCAATGATATTACTCA BHQ-1 FAM 71
Kp4 - nanK AAACAGCGGATGCTCTGAAC ATGAGTCCGCCAAGATTCTG ATAGCCTCGACCGGGATTATTCGTGA BHQ-2 TAMRA 145
Kp6 - mdtG_1 GCGTTTCAATGATCCTGTCC ATCTGCGTCTGGAAAACAG GTGCGCTGATTGGTATTGGATATGGC BHQ-2 TAMRA 76
PCR mix preparation
PCR mix preparation
PCR mix preparation for each set of primers and probe
AB
Mix Reagents Volume per well (𝛍L)
Takyon ROX Probe MasterMix dTTP blue 12.5
Forward (500 nM) 1
Reverse (500 nM) 1
Probe (250 nM) 0.5
DNA template 2.5
PCR grade water 2.5
Master Mix: TakyonTM ROX Probe MasterMix dTTP blue(Ref.UF-RPMT-B0701, Eurogentec, Belgium)

Amplification program
Amplification program

ABC
Temperature (°C) Time (minutes:seconds)
Holding stage 95.0 03:00
Cycling stage (x40) 95.0 00:15
60.0 01:00
Thermocycler: ABI StepOne Real-Time PCR system (Thermo Fischer Scientific)
Positive and negative controls
Positive and negative controls

Negative control: PCR grade water Positive controls:
  • Kp1 DNA (Klebsiella pneumoniae ATCC 13883T) at concentration 2 ng/μl ; 2.5 μl used i.e., 5 ng.
  • Kp2 DNA (Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030T)
  • Kp3 or Kp5 DNA (Klebsiella variicola subsp. variicola F2R9T; Klebsiella variicola subsp.tropica 1266T)
  • Kp4 DNA (Klebsiella pneumoniae07A044T)
  • Kp6 DNA (Klebsiella quasivariicola KPN1705T)