Detection of Bordetella pertussis and Bordetella parapertussis Using the ABI 7500 Real-time PCR System

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.

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 1mL total volume for each batch.

A	B	C	D
481-F	100	300	60
481-R	100	300	60
481-P	100	150	30
1001-F	100	150	30
1001-R	100	150	30
1001-P	100	150	30
HBG-F	100	100	20
HBG-R	100	100	20
HBG-P	100	100	20
IPC-P	100	100	20
IDTE Volume			680
Final Volume			1000

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

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.

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

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.

Vortex the thawed 20X mix, then spin down quickly before use.

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.

A	B
PCR grade water	3
Fast Advanced Master Mix	10
20X PERT Mix	1
IPC gBlock (1000 copies/uL) -added in genomics room-	1

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.

Remove the IPC gBlock and PCR gBlock controls from the -20°C freezer to thaw.

See Materials section for gBlock sequences used.

When gBlocks are fully thawed vortex and spin down briefly.

Add the IPC gBlock to the master mix cocktail as per the recipe in step 20.

Vortex the master mix and briefly spin down.

Aliquot 15µLof master mix into the required number of wells of a 96-well optical plate according to the plate map.

Order in rack and pipette in the following order:

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

2. Patient Samples and Additional NTCs:

• 5µL of each patient sample extract
• 5µLof 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:* 5µLof extract

4. HBG-IS481-pIS1001 gBlock: 5µL

5. NEC:* 5µLof 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.

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

A	B
Plate will not be run immediately	Store the 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 cycling 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
HBG	NED	MGB
IPC	CY5	None
IS481	FAM	MGB
pIS1001	VIC	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 that 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 now appear on the graph.

Under "Options" (at the bottom of the screen) Select "IS481"

Unclick Auto threshold and enter a manual value of 0.1. Click "Auto Baseline".

Repeat steps 34 & 35 for targets "pIS1001", "HBG", and "IPC"

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.

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

A	B
Any Ct value	Positive
Undetermined	Negative

A	B
35 or lower	Positive for B. pertussis or B. holmesii*
greater than 35 and less than or equal to 40	Indeterminate
Undetermined	Negative

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

A	B
35 or lower	Positive for B. parapertussis
greater than 35 and less than or equal to 40	Indeterminate
Undetermined	Negative

A	B
Any Ct value	Positive
Undetermined	Negative