TABLE 1.
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 |
STAR, stool transport and recovery.