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

Document Type: Brief Report

Authors

1 Virology Department, School of Medical Sciences, Tarbiat Modarres University, P. O. Box: 14115-111, Tehran, Iran.

2 Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran.

Abstract

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 thaninitial PCR; which can be used for diagnostic application
of HCMV in renal transplant patients.

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