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 µM of
each primers, 0.25 unit/25 µl Taq DNA polymerase,
5% DMSO, 500 µg/ml gelatin and 50-150 ng template
DNA in 25 µl 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
initial PCR; which can be used for diagnostic application
of HCMV in renal transplant patients.