Development of a semi-quantitative pcr method using a plasmid cloned with part of the gb gene of cytomegalovirus

Abstract

Cytomegalovirus (CMV) infections are highly prevalent in Brazil. CMV is a caus ative of significant morbidity and mortality in immunodepressed patients including bone marrow and kidney transplanted individuals as well as AIDS patients. The present work shows on the development of a semi-quantitative PCR for determination of CMV load in clinical samples. A 296 nucleotides segment of the gB gene of CMV was inserted into PCR II plasmids (Invitrogen, USA), and these plasmids were transfected into Escherichia coli. After confirmation of the presence of the insert and multiplication, the plasmids were quantified in the original sample. Following, a titration of the cloned plasmids was carried out in order to determinate the sensitivity of the semiquantitative PCR using primers that anneal to the gB gene of CMV. That was 867 plasmid copies/mg of DNA. The CMV load in the leukocytes of clinical samples was determined after PCR, by comparison of densities of the amplicon bands with those of the quantified cloned plasmid titration. This is a is a simple and low cost CMV semi-quantitative PCR, and it could be used for detecting viral loads in clinical samples of patients infected with CMV.