Identification of the different phylogroups from the Klebsiella pneumoniae species complex by real-time PCR
Elodie Barbier, Carla Rodrigues, Sylvain Brisse, Pascal Piveteau
Published: 2022-02-08 DOI: 10.17504/protocols.io.b4s5qwg6
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.
Steps
Primers and Probes
1.
The KpSC phylogroup ID : Primers, Probes and Amplicon length
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| 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
2.
PCR mix preparation for each set of primers and probe
| A | B |
|---|---|
| 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
3.
| A | B | C |
|---|---|---|
| **** | 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
4.
Negative control: PCR grade water
Positive controls:
- Kp 1 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 pneumoniae 07A044T)
- Kp6 DNA ( Klebsiella quasivariicola KPN1705T)
