TABLE 1.

Real-time PCR protocols for using the Roche ASRs to detect S. aureus and VRE on the LightCycler instrument

Preparation step and run conditions	Protocol to detect:
	S. aureus	mecA	VRE (vanA, vanB, and vanB2/3 genes)a
Specimen	Nasal swab	Bacterial colonies	Perianal swab
Extraction	Swab broken off into a microcentrifuge tube containing 200 μl of a 1-U/μl achromopeptidase solution	2 or 3 isolated colonies placed into a microcentrifuge tube with 1% Triton X-100, 0.5% Tween 20, 1 mmol/liter Tris-HCl (pH 8.0), and 10 mmol/liter EDTA	Swab broken off into a microcentrifuge tube containing 100 μl of STAR buffer (Roche)
Processing	Tube vortexed for 3-5 s and then incubated at 37°C for 15 min, followed by 5 min of incubation at 100°C	Tube incubated at 100°C for 10 min and then centrifuged for 1 min at >10,000 × g	Tube processed in accordance with protocol for MagNA Pure LC microbiology kit MGRADE, specimens extracted on MagNA Pure LC using the DNA MGRADE protocol
Reaction mix	2-5 μl of extracted DNA, 2 μl of LightCycler FastStart DNA master hybridization probe MGRADE mix, 2 μl of LightCycler Staphylococcus MGRADE primer/hybridization probes, 1 μl of 1:10 dilution of LightCycler Staphylococcus MGRADE recovery template, 10 μl of sterile water	2 μl of extracted DNA, 2 μl of LightCycler FastStart DNA master hybridization probe MGRADE mix, 2 μl of LightCycler mecA primer/hybridization probes, 2 μl of LightCycler mecA recovery template, 2.4 μl of MgCl2, 9.6 μl of sterile water	5 μl of extracted DNA, 2 μl of LightCycler FastStart DNA master hybridization probe MGRADE mix, 2 μl of LightCycler vanA/vanB primer/hybridization probes, 2 μl of LightCycler vanA/vanB recovery template, 2 μl of MgCl2, 7 μl of sterile water
Controls	LightCycler Staphylococcus MGRADE template set was the positive control; sterile water was the negative control	LightCycler mecA template DNA was the positive control; sterile water was the negative control	LightCycler vanA/vanB template set was the positive control; sterile water was the negative control
Real-time PCR conditions	Initial step of 10 min at 95°C, followed by amplification for 45 cycles of 10 s at 95°C, 10 s at 55°C, and 12 s at 72°C, with fluorescence acquisition at the end of each annealing	Initial step of 10 min at 95°C, followed by amplification for 45 cycles of 10 s at 95°C, 10 s at 55°C, and 12 s at 72°C, with fluorescence acquisition at the end of each annealing	Initial step of 10 min at 95°C, followed by amplification for 45 cycles of 10 s at 95°C, 10 s at 55°C, and 12 s at 72°C, with fluorescence acquisition at the end of each annealing
Melt program	Ramp to 95°C, followed by 20 s at 59°C, 20 s at 45°C at a rate of 0.2°C/s, and a gradual increase to 85°C at a rate of 0.2°C/s with continuous fluorescence acquisition	Ramp to 95°C, followed by 20 s at 59°C, 20 s at 45°C at a rate of 0.2°C/s, and a gradual increase to 85°C at a rate of 0.2°C/s with continuous fluorescence acquisition	Ramp to 95°C, followed by 20 s at 59°C, 20 s at 45°C at a rate of 0.2°C/s, and a gradual increase to 85°C at a rate of 0.2°C/s with continuous fluorescence acquisition

^aSTAR, stool transport and recovery.