PCR optimization: Improving of Human Cytomegalovirus (HCMV) PCR to Achieve a Highly Sensitive Detection Method

Abstract

Polymerase chain reaction (PCR) is a rapid and simple technique with high sensitivity and specificity. In<br />the recent years, PCR has been used for rapid detection of viral nucleic acids, such as Human<br />cytomegalovirus (HCMV), whereas, PCR optimization is an important task to be done, especially before<br />it's diagnostic application. Annealing temperature, ion concentration (especially Mg2+ ion) and the<br />cycling program and enhancer compounds are important optimization parameters. Peripheral blood<br />leukocytes (PBLs) were isolated from samples collected from renal transplant recipients suffering from<br />severe and symptomatic CMV disease. PBLs DNA was extracted and used for PCR. Annealing temperature<br />and MgCl2 concentration and cycling condition were optimized. Dimethyl sulfoxide (DMSO) and gelatin<br />were checked as enhancer components. The optimized condition obtained through this study was:<br />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 of<br />each primers, 0.25 unit/25 μl Taq DNA polymerase, 5% DMSO, 500 μg/ml gelatin and 50-150 ng template<br />DNA in 25 μl final volume. PCR was performed as: 95°C 5 min (pre-denaturation), 94°C 50 sec, 58°C<br />1 min, 72°C 1 min for 35 cycles and 72°C 5 min (final extension). Using these conditions, it was shown that<br />optimized PCR was five fold more sensitive thaninitial PCR; which can be used for diagnostic application<br />of HCMV in renal transplant patients.<br /><br />