PCR optimization: Improving of human cytomegalovirus (HCMV) PCR to achieve a highly sensitive detection method†

Polymerase chain reaction (PCR) is a rapid and simple technique with high sensitivity and specificity. In the recent years, PCR has been used for rapid detection of viral nucleic acids, such as Human cytomegalovirus (HCMV), whereas, PCR optimization is an important task to be done, especially before it’s diagnostic application. Annealing temperature, ion concentration (especially Mg2+ ion) and the cycling program and enhancer compounds are important optimization parameters. Peripheral blood leukocytes (PBLs) were isolated from samples collected from renal transplant recipients suffering from severe and symptomatic CMV disease. PBLs DNA was extracted and used for PCR. Annealing temperature and MgCl2 concentration and cycling condition were optimized. Dimethyl sulfoxide (DMSO) and gelatin were checked as enhancer components. The optimized condition obtained through this study was: 1x PCR buffer (20 mM Tris-HCl pH 8.6, 50 mM KCl), 2.5 mM MgCl2, 0.2 mM of each dNTPs, 0.25 ىM of each primers, 0.25 unit/25 ىl Taq DNA polymerase, 5% DMSO, 500 ىg/ml gelatin and 50-150 ng template DNA in 25 ىl final volume. PCR was performed as: 95°C 5 min (pre-denaturation), 94°C 50 sec, 58°C 1 min, 72°C 1 min for 35 cycles and 72°C 5 min (final extension). Using these conditions, it was shown that optimized PCR was five fold more sensitive than † This study was presented at the second National Biotechnology Congress, Islamic Republic of Iran, Oct. 9-11, 2001, Karaj, Iran initial PCR; which can be used for diagnostic application of HCMV in renal transplant patients.