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.

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 1mLtotal volume for each batch.

A	B	C	D
bfrZ-Qp (FAM) (Bronch-P)	100	100	20
bfrZ-Qf-mod (Bronch-F)	100	100	20
bfrZ-Qr (Bronch-R)	100	100	20
BHIS62U28P (CY5) (Holm-P)	100	150	30
BHIS41U20 (Holm-F)	100	300	60
BHIS91L17 (Holm-R)	100	300	60
IDTE Volume			790
Final Volume			1000

Pipette the 20X Holm mix into 100µL aliquots. Label each aliquot with 20X Holm Mix.

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.

Turn on the BSC in the Reagent Preparation Clean Room. Allow the BSC to stabilize, and decontaminate before use.

Remove an aliquot of the 20X Holm Mix from its place of storage.

If frozen let thaw completely before using.

When the 20X mix is completely thawed vortex briefly and spin down.

In a 1.7 mL microcentrifuge tube prepare the master mix cocktail as follows:

A	B
PCR Grade Water	4
Fast Advanced Master Mix	10
20X Holm Mix	1

Make enough master mix to account for the total number of reactions on your PCR plate + 15% to account for pipetting errors.

Vortex and briefly spin down the master mix before pipetting

Aliquot 15µL of master mix into the required number of wells of a 96-well optical plate

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.

Turn on the BSC in a Genomic PCR Room. Allow the BSC to stabilize, ensure it is functioning as expected and decontaminate before use.

Pipette samples from the sample extract plate and controls in the following order:

1. NTC: 5µL of the same lot PCR grade water that was used to prepare the master mix.

2. Patient Samples: 5µL of each sample extract from the extract plate

3. HOLM/BRONCH (bfrZ-hIS1001) gBlock: 5µL

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

A	B
Plate will not be run immediately	Store plate in a 4C fridge until ready to perform PCR
Plate will be run immediately	Proceed to load and run the plate on the ABI 7500

On the ABI 7500 instrument create a new experiment for the current run as per the ABI 7500 User Manual

Check that the following reaction conditions are correctly programmed:

A	B	C
50	2 min	Hold
95	20 sec	Hold
95	3 sec	40
60	30 sec

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.

Check that each sample well has the correct target, dye, and quencher assigned to it.

A	B	C
bfrZ (Bronch)	FAM	None
hIS1001 (Holm)	CY5	None

Load the plate onto the ABI 7500 instrument.

Save the run on the instrument and start the run as per the ABI 7500 user manual.

Ensure the run has completed successfully. Click OK.

Select the Analysis tab after the run has completed, and then select the "Amplification Plot" tab.

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.

Under Options (at the bottom of the screen), select "hIS1001" ( B. holmesii )

Unclick "Auto Threshold" and enter a manual value of 0.1 in the box. Click Auto Baseline.

Repeat steps 25 and 26 but for the target "bfrZ" ( B. bronchiseptica )

Click on the "Analyze" button.

Ensure that under "Plot Settings" the "Delta Rn vs. Cycle" option is selected and plot colour is set to "target".

View the results of all run controls and ensure they have produced acceptable values before proceeding with clinical sample analysis.

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.

A	B	C	D	E
POS	NEG	NEG	NEG	B. pertussis Positive
POS/NEG	NEG	POS	NEG	B. holmesii Positive
POS/NEG	POS/NEG	NEG	POS	B. bronchiseptica Positive
NEG	POS	NEG	NEG	B. parapertussis Positive