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. Inthe recent years, PCR has been used for rapid detection of viral nucleic acids, such as Humancytomegalovirus (HCMV), whereas, PCR optimization is an important task to be done, especially beforeit’s diagnostic application. Annealing temperature, ion concentration (especially Mg2+ ion) and thecycling program and enhancer compounds are important optimization parameters. Peripheral bloodleukocytes (PBLs) were isolated from samples collected from renal transplant recipients suffering fromsevere and symptomatic CMV disease. PBLs DNA was extracted and used for PCR. Annealing temperatureand MgCl2 concentration and cycling condition were optimized. Dimethyl sulfoxide (DMSO) and gelatinwere 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 μM ofeach primers, 0.25 unit/25 μl Taq DNA polymerase, 5% DMSO, 500 μg/ml gelatin and 50-150 ng templateDNA in 25 μl final volume. PCR was performed as: 95°C 5 min (pre-denaturation), 94°C 50 sec, 58°C1 min, 72°C 1 min for 35 cycles and 72°C 5 min (final extension). Using these conditions, it was shown thatoptimized PCR was five fold more sensitive thaninitial PCR; which can be used for diagnostic applicationof HCMV in renal transplant patients.