TITLE: Detection of Bordetella pertussis and Bordetella parapertussis Using the ABI 7500 Real-time PCR System 
AUTHORS: Martin Cheung, Tracy Lee, Robert B Azana, Loretta Janz, Natalie Prystajecky, Linda Hoang

[DESCRIPTION]
This procedure provides instructions for how to perform qualitative PCR (qPCR) for the detection of the IS481 insertion sequence of Bordetella pertussis and the pIS1001 insertion sequence of the Bordetella parapertussis from appropriate direct specimens. The IS481 insertion has been found to be present in some strains of B. bronchiseptica and most strains of B. holmesii. Some strains of B. bronchseptica also carry the pIS1001insertion sequence. The B. holmesii and B. bronchiseptica multiplex qPCR should be performed when indicated.

[GUIDELINES]
Perform all manipulations of samples and DNA in a genomic level PCR laboratory. Prepare PCR master mixes in a Reagent Preparation Clean Room. 

[STEPS]
SECTION: Procedure A: Preparing 20X PCR Master Mix
1. All primer and probe sequences are listed in the materials section of this protocol. 

Order all necessary primers and probes; if received lyophilized reconstitute as per the manufacturers instructions before making desired stock dilutions. 
SECTION: Procedure A: Preparing 20X PCR Master Mix
2. Turn on the Biological Safety Cabinet (BSC) in the Reagent Preparation Clean Room. Allow the BSC to stabilize, ensure it is functioning as expected, and decontaminate prior to using.  

Inside the BSC, prepare 20X PERT Mix as per the following table. Prepare 1 mL  total volume for each batch. 

 
 

SECTION: Procedure A: Preparing 20X PCR Master Mix
3. Pipette the 20X PERT mix into 100 µL aliquots. Label each aliquot with 20X PERT Mix.

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
5. Turn on the BSC in the Reagent Preparation Clean Room. Allow the BSC to stabilize, and decontaminate before use. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
6. Remove an aliquot of 20X PERT Mix from the -20 °C freezer and allow to thaw.

Note: probes are light sensitive - store away from light.

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
7. Vortex the thawed 20X mix, then spin down quickly before use. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
8. In a 1.7 mL microcentrifuge tube prepare the master mix cocktail as follows, ensuring all reagents are mixed thoroughly before pipetting into the 96-well plate. 

Add the first 3 reagents in a Reagent Preparation Clean Room. The IPC gBlock is stored in the PCR Genomic Room and is added after transfer to that room.
 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
9. In a clear plastic bag, transfer the microcentrifuge tube with the first 3 ingredients of the master mix from the Reagent Preparation Clean Room to the PCR Genomic Room. 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
10. Remove the IPC gBlock and PCR gBlock controls from the -20 °C freezer to thaw. 

See Materials section for gBlock sequences used.  

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
11. When gBlocks are fully thawed vortex and spin down briefly. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
12. Add the IPC gBlock to the master mix cocktail as per the recipe in step 20. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
13. Vortex the master mix and briefly spin down.  
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
14. Aliquot 15 µLof master mix into the required number of wells of a 96-well optical plate according to the plate map. 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
15. Order in rack and pipette in the following order:

1. Initial NTC: 5 µLof the same lot PCR grade water that was used to prepare the master mix. 
2. Patient Samples and Additional NTCs: 
5 µL of each patient sample extract
 5 µLof additional NTCs of the same lot of PCR grade water that was used to prepare the master mix. Run approximately every 15th well. 
3. PEC*: 5 µLof extract
4. HBG-IS481-pIS1001 gBlock: 5 µL 
5. NEC*: 5 µLof extract 


Note*: It is suggested to use a positive and negative extraction control as per individual laboratory practice. Please see "Controls" in the Materials section for more details on the PEC and NEC. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
16. Apply an optical 96-well plate film to the plate using the plate seal applicator, taking care to seal the plate edges and avoid touching the top of the film. 

Note: any fingerprints or residue on the film will alter the optical readings 


 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
17. On the ABI 7500 instrument create a new experiment for the current run as per the ABI 7500 User Manual.  

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
18. Check that the following cycling conditions are correctly programmed: 


 

SECTION: Procedure A: Preparing 20X PCR Master Mix
4. Store 20X PERT aliquots in 4 °C fridge for short-term storage
and a -20 °C freezer inside a Reagent Preparation Clean Room for long-term storage. 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
20. Check that each sample well has the correct target, dye, and quencher assigned to it. 


 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
19. Either input your samples and controls into the current run file manually or import the data from a saved run file on a memory stick. 

Ensure all wells are assigned correctly on the run file as per the pipetting of the sample plate. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
21. Load the plate onto the ABI 7500 instrument.
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
22. Save the run on the instrument and start the run as per the ABI 7500 user manual. 
SECTION: Procedure D: 7500 FAST Run Analysis
23. Ensure that the run has completed successfully. Click OK. 

SECTION: Procedure D: 7500 FAST Run Analysis
24. Select the Analysis tab after the run has completed, and then select the "Amplification Plot" tab. 

    
SECTION: Procedure D: 7500 FAST Run Analysis
26. Under "Options" (at the bottom of the screen) Select "IS481"
SECTION: Procedure D: 7500 FAST Run Analysis
27. Unclick Auto threshold and enter a manual value of 0.1. Click "Auto Baseline". 
SECTION: Procedure D: 7500 FAST Run Analysis
28. Repeat steps 34 & 35 for targets "pIS1001", "HBG", and "IPC" 
SECTION: Procedure D: 7500 FAST Run Analysis
29. Click on the "Analyze" button. 
SECTION: Procedure D: 7500 FAST Run Analysis
30. Ensure that under "Plot Settings" the "Delta Rn vs. Cycle" option is selected and plot colour is set to "target".  
SECTION: Procedure D: 7500 FAST Run Analysis
31. View the results of all run controls and ensure they have produced acceptable values before proceeding with clinical sample analysis.  
SECTION: Procedure D: 7500 FAST Run Analysis
32. Clinical Samples:

Examine results for the HBG, IS481, pIS1001, and IPC targets for each clinical sample. 

Note: All Ct values must be confirmed by viewing the amplification curve and multicomponent plot for appropriate graphing of positive result. 

See the Applied Biosystems 7500 Fast Real-Time PCR System Presence/Absence Experiments manual at: 

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_050341.pdf



 

 
* Please see method limitation #1 in the Warning section. 


 


 


SECTION: Procedure D: 7500 FAST Run Analysis
25. To view all samples, click on the small square at the top left of the plate map to select all the wells on the plate at once. Curves should now appear on the graph.