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PCR conditions were one cycle of 5 min at 94°C, 40 cycles of 1 min at 94°C, 1 min 20 s at 59.5°C, and 2 min at 72°C, followed by a final cycle of 7 min at 72°C.

PCR cycling conditions were 95°C for 5 min followed by 30 cycles of 95°C for 30s, 55°C for 30s, 72°C for 1.5 min followed by a final cycle of 72°C for 10 min. All urine gDNAs yielded robust amplicons after 30 PCR cycles, whereas identically processed mock specimens (molecular grade water) failed to yield amplicons after 40 cycles.

This profile was preceded by an initial cycle with a 5-min denaturation at 94°C, and it was followed by a final cycle with a 7-min extension step at 72°C.

The cycling parameters were as follows: an initial denaturation of 96°C for 3 min followed by 30 cycles of 96°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min and followed by a final cycle of 72°C for 4 min.

The condition for the PCR reaction was 94°C for 1 min for initial denaturation, followed by 25 cycles of denaturing at 94°C for 15 sec, annealing at 55°C for 30 sec, and extension at 60°C for 60 sec, followed by a final cycle of extension for 10 min. The reagents were PCR kit from Clonetech (Palo Alto, CA).

The thermocycler protocol used to amplify bisulfite-modified DNA was as follows: 5 min at 95°C, 50 repeats at 95°C for 1 min, followed by 60°C for 1 min, followed by 72°C for 1 min, which was then followed by a final cycle of 5 min at 72°C and then terminated at 4°C.

Twenty-four additional cycles of PCR were then performed as above but each with an annealing temperature of 56°C followed by a single final cycle of 10 min at 72°C.

PCR was completed by an additional final cycle at 72°C for 30 sec.

PCR conditions used the following profile: 94°C for 4 minutes (one cycle); one minute each of 94°C, 50°C and 72°C (35 cycles), followed by a final extension cycle of 72°C for 5 minutes.

Polymerase chain reaction (PCR) products were amplified using Bioline Taq (Bioline, London, UK) in the following conditions: 5 min 96°C initial denaturation, 96°C 30 s, 55°C 1 min, 72°C 30 s for 30 cycles, followed by a final extension cycle for 5 min at 72°C (Hybaid, Ashford, UK).

Next, 100 μl of probe was injected onto the slide and hybridised with agitation at 50°C for 16 hours, followed by sequential washing in four cycles of 2× SSC, 0.1% SDS, 2 cycles of 1× SSC, 0.1% SDS, and 0.1× SSC, 0.2% SDS at 40°C, 2 cycles of 0.1× SSC at 23°C, followed by a final cleaning cycle of ultrapure water before drying.