The tube containing the reaction mixture was placed in a preheated (94 C) thermal cycler.
The first-stage PCR profile was: 1 94 C for 1 min followed by 50 C for 3 min, and 72 C for 1 min. The second-stage PCR amplification profilewas as follows: 40 94 C for 40 s, followed by 65 C for 40 s, 72 C for 40 s, and a 5 min final extension at 72 C. The
PCR products amplified by 20 ACP primers were resolved in a 2% agarose gel, stained with ethidium bromide, and visualized under UV and documented in a Kodak Gel Logic200 system (Carestream Health Inc, Rochester, NY).