Nov 05, 2024
Real-time PCR amplification and genotyping of the 23s rRNA from Bordetella pertussis using the LightCycler 480
- Valérie Bouchez1,2,
- Sylvain Brisse1,2,
- Carla Rodrigues1,2
- 1Institut Pasteur, French NRC for Whooping cough and other Bordetella infections;
- 2Institut Pasteur, Biodiversity and Epidemiology of Bacterial Pathogens unit
Protocol Citation: Valérie Bouchez, Sylvain Brisse, Carla Rodrigues 2024. Real-time PCR amplification and genotyping of the 23s rRNA from Bordetella pertussis using the LightCycler 480. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q4y1v25/v1
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: November 04, 2024
Last Modified: November 05, 2024
Protocol Integer ID: 111520
Keywords: macrolide resistance, 23s rRNA, mutation, qPCR, Bordetella pertussis
Abstract
The method is an adaptation for LC480 (version II, Roche) of the one described by Kamachi et al., EID 2020 (PMID: 32946738 - Supplementary Materials "Cycleave Real-Time PCR Assay for Detection of the A2047G Mutation"). qPCR targeting 23S rRNA differentiates two alleles of the ribosomal RNA of the 23S subunit of Bordetella pertussis (present in 3 copies per genome). The A2047G mutation distinguishes macrolide-susceptible isolates, with an A at position 2047, from macrolide-resistant Bordetella pertussis isolates (MRBP), with a G at position 2047.
Real-time amplification is performed from DNA extracted from a respiratory sample that has previously given a positive result for the detection of pertussis toxin (qPCR PTa, 1 copy per genome). Two distinct fluorescent probes are employed: one specific for the susceptible allele (FAM) and the other for the resistant allele (HEX). These probes specifically detect a 123 bp fragment of the 23S ribosomal RNA of B. pertussis and identify the A2047G mutation.
This qPCR is performed only for samples that have given a positive qPCR-PT result, which has a similar analytical sensitivity.
Attachments
Guidelines
This qPCR is performed only for samples that have given a positive qPCR-PT result, which has a similar analytical sensitivity.
Materials
EQUIPMENT AND MATERIALS
- Sterile 1.5 mL Eppendorf tubes
- LightCycler 480 Multiwell plate 96, Roche, ref. no. 04 729 692 001
- Centrifuge for 1.5 mL Eppendorf tubes and centrifuge for microplates
- PCR refrigerated racks
- LightCycler 480 Thermal Cycler Version II, Roche
REAGENTS
- DNAaway, MBP, Ref. 038188
- LightCycler 480 Probes Master Kit, Roche, ref. no. 04 707 494 001
- Water, molecular biology quality, supplied with kit Ref. 04 707 494 001 or by default Sterile Distilled H2O (10 mL ampoule, Braun Medical B, Ref. 3511810)
POSITIVE OR STANDARD CONTROLS
Genomic DNA extracted from the reference strain Tohama (CIP 8132), either using the QIAGEN "DNeasy Blood & Tissue Kit (A69504)" or via the MAXWELL automatic extractor with the "Maxwell‱ CSC Blood DNA Kit (AS1321)". This DNA serves as a susceptible positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C. The tube is identified by the name of the strain and the date of aliquoting of the batch.
Genomic DNA extracted from the FR4991 strain (CIP 112499), either using the QIAGEN DNeasy Blood & Tissue Kit (A69504) or via the MAXWELL Automatic Extractor with the Maxwell‱ CSC Blood DNA Kit (AS1321). This DNA serves as a resistant positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C.
PRIMERS AND PROBES (HPLC quality, supplier TibMolBiol, Berlin, Germany)
They are supplied freeze-dried for "long-term" storage, protected from light, between +2°C and +8°C. Probes and primers are resuspended in H2O, molecular biology quality, and can be stored between -15°C and -25°C. Refer to the supplier's data sheets for quantities per tube.
Primers: 23S-rRNA-F and 23S-rRNA-R
23S-rRNA-F: 5'-GAATGGCGTAACGATG -3'
23S-rRNA-R: 5'- TGCAAAGCTACAGTAAAGG -3'
Probes:
23s_rRNA_SUS: 6FAM-CGGCTAGACGGAAAGACCCCA-BHQ2
23s_rRNA_RES: HEX-CGGCTAGACGGGAAGACCCCA-BHQ2
Safety warnings
POSITIVE OR STANDARD CONTROLS
Genomic DNA extracted from the reference strain Tohama (CIP 8132), either using the QIAGEN "DNeasy Blood & Tissue Kit (A69504)" or via the MAXWELL automatic extractor with the "Maxwell‱ CSC Blood DNA Kit (AS1321)". This DNA serves as a susceptible positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C. The tube is identified by the name of the strain and the date of aliquoting of the batch.
Genomic DNA extracted from the FR4991 strain (CIP 112499), either using the QIAGEN DNeasy Blood & Tissue Kit (A69504) or via the MAXWELL Automatic Extractor with the Maxwell‱ CSC Blood DNA Kit (AS1321). This DNA serves as a resistant positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C.
These strains (Tohama and FR4991) are deposited in the Institut Pasteur collection (CIP 8132 and CIP 112499, respectively) – https://catalogue-crbip.pasteur.fr/recherche_catalogue.xhtml – and can be ordered directly from their website.
QUALITY ASSURANCE
When the batch of the LightCycler 480 Probes Master Kit (Roche, Art. No. 04 707 494 001) expires, it can still be used as long as it is systematically revalidated with the positive amplification controls.
PRIMERS AND PROBES
PRIMERS AND PROBES
They are supplied freeze-dried for "long-term" storage, protected from light, between +2°C and +8°C. Probes and primers are resuspended in H2O, molecular biology quality, and can be stored between -15°C and -25°C. Refer to the supplier's data sheets for quantities per tube.
Primers:
23S-rRNA-F: 5'-GAATGGCGTAACGATG -3'
23S-rRNA-R: 5'- TGCAAAGCTACAGTAAAGG -3'
Probes:
23s_rRNA_SUS: 6FAM-CGGCTAGACGGAAAGACCCCA-BHQ2
23s_rRNA_RES: HEX-CGGCTAGACGGGAAGACCCCA-BHQ2
SAMPLE PROCESSING
SAMPLE PROCESSING
DNA from respiratory samples is extracted according to the protocol "Genomic DNA extraction with the High Pure PCR Template Preparation Kit (ROCHE) for use with the Bordetella R-gene and Parapertussis R-gene kits".
EXPERIMENTAL PROTOCOL
EXPERIMENTAL PROTOCOL
Plate Plan
The plate design must include:
- a negative amplification control (H2O tube, Roche kit)
- a negative H2O extraction control, if DNA extraction carried out in the lab
- positive amplification control for susceptible B. pertussis: CIP 8132 at 100 pg/μL and 10 pg/μL
- positive amplification control for resistant B. pertussis: CIP 112499 at 100 pg/μL and 10 pg/μL
- samples to test (case n°1)
Case n°1
For a respiratory sample received already extracted or extracted with the procedure "Genomic DNA extraction by the High Pure PCR Template Preparation Kit (ROCHE) for use with the Bordetella R-gene and parapertussis R-gene kits", add 5 μL of the extracted DNA to the dedicated well (according to the plate plan).
Preparation of the of the Assay Mix 20X
In the reaction mix preparation room prepare the assay mix 20X
| A | B | |
| Reagent (final concentration) | Volume | |
| H2O | 56 μL | |
| Primer 23srRNA-F (18 μM) | 18 μL | |
| Primer 23srRNA-R (18 μM) | 18μL | |
| 23s_rRNA_SUS (4 μM) | 4 μL | |
| 23s_rRNA_RES (4 μM) | 4 μL | |
| Total volume | 100 μL |
- Mix gently by aspiration/discharge with a pipette.
- Storage between -15°C and -25°C (maximum 12 months).
Preparation of the reaction mixture (volumes are given for one sample)
- Prepare the reaction mixture (by multiplying the quantity in the "Volume" column by the number of reactions to be performed, plus two additional reactions).
In a 1.5 mL Eppendorf tube:
| A | B | C | |
| Reagent | Volume | Final Concentration | |
| H2O | 4 μL | ||
| Assay Mix 20X | 1 μL | 1X | |
| Probes Master 2X | 10 μL | 1X | |
| Total volume | 15 μL |
- Mix gently by aspiration/discharge with a pipette.
- Place the 96-well plate in the refrigerated rack.
- Dispense 15 μL of the reaction mixture into the wells following the plate plan.
- Cover the plate with aluminum foil to get out of the room.
- Don't forget to use the adhesive to seal the plaque later.
Reminder
When the reaction mix preparation part comes out, place the plate in the dedicated refrigerated rack.
At a DNA sample repository:
- Add 5 μL of extracted and control DNA to the wells according to the plate plan.
- Immediately cover the plate with the appropriate adhesive.
In PCR room where the thermal cycler is located:
- Centrifuge the plate for 1 minute at about 1000 g at room temperature.
- Put the plate in the LightCycler 480 and start the "qPCR 23s rRNA" program
Amplification Program
DATA ANALYSIS
DATA ANALYSIS
The analysis of the bacterial target is performed in "Endpoint Genotyping" mode, with FAM (465-510 nm filter) and HEX (533-580 nm filter) readout.
In the "Sample Editor" tab, negative and extraction controls should be set as "Negative Control", and positive controls as "Standards" in the "EndPt Sample Type" column.
- For standards, add "susceptible" for CIP 8132 at 10 and 100 pg/μL to the "EndPt Genotype" column, and "resistant" for FR4991 at 10 and 100 pg/μL.
- The samples to be tested must be set as "Unknown" in the "Sample Type" column.
- Finally, carry out the analysis.
Example:
The final conclusion for a sample will be determined based on the alleles called. In general, the "endpoint fluorescence" for a susceptible sample is always higher for the susceptible allele (FAM) than for the resistant allele (HEX), and vice versa for a resistant sample.
It is important to note that for samples with cycle thresholds (Ct) greater than 35 in the PTa qPCR, the majority will be categorized as negative in the qPCR 23S rRNA PCR.
In all cases, the final decision on the interpretation of the results is taken by the NRC (deputy) head, who are also responsible for the diagnosis made.
POSITIVE OR STANDARD CONTROLS
POSITIVE OR STANDARD CONTROLS
Genomic DNA extracted from the reference strain Tohama (CIP 8132), either using the QIAGEN "DNeasy Blood & Tissue Kit (A69504)" or via the MAXWELL automatic extractor with the "Maxwell‱ CSC Blood DNA Kit (AS1321)". This DNA serves as a susceptible positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C. The tube is identified by the name of the strain and the date of aliquoting of the batch.
Genomic DNA extracted from the FR4991 strain (CIP 112499), either using the QIAGEN DNeasy Blood & Tissue Kit (A69504) or via the MAXWELL Automatic Extractor with the Maxwell‱ CSC Blood DNA Kit (AS1321). This DNA serves as a resistant positive control and is used at the concentration of 100 pg/μL and 10 pg/μL. It is aliquoted to be stored between -15°C and -25°C.
Protocol references
Kamachi K, Duong HT, Dang AD, Hai T, Do D, Koide K, Otsuka N, Shibayama K, Hoang HTT. Macrolide-Resistant Bordetella pertussis, Vietnam, 2016-2017. Emerg Infect Dis. 2020 Oct;26(10):2511-2513. doi: 10.3201/eid2610.201035. PMID: 32946738; PMCID: PMC7510698.