TITLE: Detection of Bordetella holmesii and Bordetella bronchiseptica 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 hIS1001 insertion sequence of Bordetella holmesii and the bfrZ gene of Bordetella bronchiseptica from appropriate direct specimens. This test is intended to be used as a secondary assay to differentiate B. pertussis (IS481 positive) from B. holmesii. 

[GUIDELINES]
Perform all manipulations of samples and DNA in a genomic level PCR laboratory. Prepare all 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 the 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 HOLM mix as per the following table. Prepare 1 mLtotal volume for each batch. 


 




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

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

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 the 20X Holm Mix from its place of storage. 

If frozen let thaw completely before using. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
7. When the 20X mix is completely thawed vortex briefly and spin down. 
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:

 
 Make enough master mix to account for the total number of reactions on your PCR plate + 15% to account for pipetting errors. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
9. Vortex and briefly spin down the master mix before pipetting
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
10. Aliquot 15 µL of master mix into the required number of wells of a 96-well optical plate 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
11. Seal the 96-well optical plate with adhesive film using a seal applicator and place in a clear plastic bag for transport to a Genomic PCR Room. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
12. Turn on the BSC in a Genomic PCR Room. Allow the BSC to stabilize, ensure it is functioning as expected and decontaminate before use.  
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
13. Pipette samples from the sample extract plate and controls in the following order:

1. NTC: 5 µL of the same lot PCR grade water that was used to prepare the master mix. 
2. Patient Samples: 5 µL of each sample extract from the extract plate
3. HOLM/BRONCH (bfrZ-hIS1001) gBlock: 5 µL 


SECTION: Procedure B: Setting up the Real-Time PCR Reactions
14. Apply an optical adhesive film to the plate using a 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
15. 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
16. Check that the following reaction conditions are correctly programmed:


 

SECTION: Procedure B: Setting up the Real-Time PCR Reactions
17. 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
18. 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. Load the plate onto the ABI 7500 instrument. 
SECTION: Procedure B: Setting up the Real-Time PCR Reactions
20. Save the run on the instrument and start the run as per the ABI 7500 user manual. 
SECTION: Procedure C: 7500 FAST Run Analysis
21. Ensure the run has completed successfully. Click OK. 


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


SECTION: Procedure C: 7500 FAST Run Analysis
23. 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 appear on the graph. 
SECTION: Procedure C: 7500 FAST Run Analysis
24. Under Options (at the bottom of the screen), select "hIS1001" (B. holmesii)
SECTION: Procedure C: 7500 FAST Run Analysis
25. Unclick "Auto Threshold" and enter a manual value of 0.1 in the box. Click Auto Baseline.
SECTION: Procedure C: 7500 FAST Run Analysis
26. Repeat steps 25 and 26 but for the target "bfrZ" (B. bronchiseptica)
SECTION: Procedure C: 7500 FAST Run Analysis
27. Click on the "Analyze" button. 
SECTION: Procedure C: 7500 FAST Run Analysis
28. Ensure that under "Plot Settings" the "Delta Rn vs. Cycle" option is selected and plot colour is set to "target". 
SECTION: Procedure C: 7500 FAST Run Analysis
29. View the results of all run controls and ensure they have produced acceptable values before proceeding with clinical sample analysis. 
SECTION: Procedure C: 7500 FAST Run Analysis
30. Examine the results for hIS1001 and bfrZ 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

Interpretation Table
POS: Ct < or = 35
NEG: Ct = Undetermined
If Ct >35 sample is indeterminate and may require retesting or recollection.