TITLE: Live/Dead qPCR of B. pertussis IS481
AUTHORS: Iain MacArthur, Stacy Ramkissoon, Andrew Preston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the use of qPCR for the quantitative determination of live </span><span style = "font-style:italic;">B. pertussis</span><span> bacteria in a sample. The method uses propidium monoazide, a compound that can cross the cell wall of dead bacteria and binds DNA following photo activation to inhibit amplification of DNA from dead bacteria. This method uses a TaqMan probe specific to IS481 to ensure maximum sensitivity due to the high copy number of the target. Although IS481 is not specific to </span><span style = "font-style:italic;">B. pertussis </span><span>for experimental purposes it provides acceptable specificity. </span></div></div>

[STEPS]
?. [PMA treatment]
Pellet fresh samples by centrifuging at 2,000 x g for 10 minutes and resuspend in 1.2 ml of PBS.
?. [PMA treatment]
Transfer 200µl of resuspended sample into two clear microfuge tubes (continue with 1 sample to Step #3 and 1 sample skip to Step #4).
?. [PMA treatment]
Add 0.5µl of PMA to one of the 200µl of samples from Step #2.
?. [PMA treatment]
Incubate microfuge tubes in the dark for 10 min at room temperature. Cover samples with aluminium foil and incubate on a rocker.
?. [PMA treatment]
Expose samples to light using the PMA-Lite™ LED Photolysis Device for 5 min.
?. [DNA purification]
Add 20µl of QIAGEN Protease and 4 µl of RNase A.
?. [DNA purification]
Add 200µl of Buffer AL and vortex for 15 s.
?. [DNA purification]
Incubate at 56ºC for 10 min.
?. [DNA purification]
Briefly spin tube to recover condensation.
?. [DNA purification]
Add 200µl of ethanol (96-100%), vortex and spin briefly.
?. [DNA purification]
Add to spin column and centrifuge for 1 min at 6000xg.
?. [DNA purification]
Transfer to a new collection tube.
?. [DNA purification]
Add 500µl AW1 buffer and spin for 1 min at 6000xg.
?. [DNA purification]
Transfer to a new collection tube.
?. [DNA purification]
Add 500µl AW2 buffer and spin for 3 min at 17000xg
?. [DNA purification]
Transfer to a 2ml microfuge tube.
?. [DNA purification]
Spin at max speed for 1 min.
?. [DNA purification]
Transfer to a 1.5ml microfuge tube.
?. [DNA purification]
Add 200µl of Buffer AE and incubate at room temperature for 1 min.
?. [DNA purification]
Elute by centrifuging for 1 min at 6000xg.
?. [qPCR]
Dilute stock primers (50µM) 9 in 50µl, to give a reaction concentration of 900nm.
?. [qPCR]
Dilute stock probe (50µM) 3 in 100µl, to give a reaction concentration of 150nm.
?. [qPCR]
Prepare Mastermix as follows:  ABC1Taqman MM10µl1x2Forward primer2µl900nM3Reverse primer2µl900nM4Probe2µl150nM
ABC1Taqman MM10µl1x2Forward primer2µl900nM3Reverse primer2µl900nM4Probe2µl150nM
?. [qPCR]
Add 16µl of master mix per well.
?. [qPCR]
Serial dilute 9 times, 5:50 positive control DNA (25ng/ul).
?. [qPCR]
Use dilutions 3-9 for 1000, 100, 10, 1, 0.1, 0.01, 0.001pg.
?. [qPCR]
Load in triplicate 4µl of DNA for each control dilution, unknown and water samples.
?. [PCR and analysis]
Cover plate with film and spin for 1 min at 1000xg
?. [PCR and analysis]
Set up qPCR program as follows:  ABC150ºC2min295ºC10min340 cycles:95ºC15sec460ºC1min
ABC150ºC2min295ºC10min340 cycles:95ºC15sec460ºC1min
?. [Results]
Run analysis on data using StepOnePlus™ Software v2.3.
?. [Results]
Outliers of triplicate samples should be ignored (omit).
?. [Results]
Confirm standard curve has an r2 >0.95.
?. [Results]
Convert pg of DNA to copy number:  B. pertussis/sample=(50x(mass of template in pg)x6.022x1023)/length of genome in bp*1x1012x650).
?. [Results]
Untreated sample (no  PMA), represents total B. pertussis in the sample.
?. [Results]
PMA treated sample gives number of live B. pertussis in the sample.
?. [Results]
The number of dead can be determined from the difference between untreated and treated.