Quantitation of human cytomegalovirus DNA in leukocytes by end-point titration and duplex polymerase chain reaction

Abstract

The presence of human cytomegalovirus (CMV) DNA and cellular DNA in leukocytes was detected by duplex polymerase chain reaction (PCR) and quantitated by end-point titration. Two different duplex PCR methods were used to co-amplify CMV DNA and a 536 bp fragment of globin DNA. MIE-globin PCR amplified a 435 bp fragment of the major immediate early (MIE) gene of CMV DNA whereas the LA-globin PCR amplified a 200 bp fragment of the late antigen (LA) gene of CMV DNA. PCR products were separated by electrophoresis in 3% agarose gels and detected by ethidium bromide staining. Amplification of globin DNA was included in the PCR as a positive control to monitor the accuracy and reproducibility of the PCR assay and to provide a reference point for CMV DNA levels. End-point titration PCR using known amounts of recombinant CMV DNA and human placental DNA showed that the end-point titres of the amplified CMV DNA correlated directly with the amount of CMV DNA in the sample. The limit of detection of MIE-globin and LA-globin PCR was 1 ng for placental DNA, and 10 fg (1000 copies) for CMV-MIE DNA and 1 fg (100 copies) for CMV-LA DNA, respectively. The amount of CMV DNA was quantitated in leukocytic lysates of 16 immunocompromised patients, who were tested for the presence of CMV in blood by cell culture, and of four normal controls. The blood concentration of CMV DNA, calculated as the number of copies of CMV DNA per μg of leukocyte DNA, varied between 10 4 and 10 7 in the seven bloods that were CMV-cell-culture-positive, and between 10 2 and 10 4 in the blood of five patients that were CMV-cell-culture-negative. CMV DNA was undetected by PCR in the blood of another eight CMV-negative cases. This study shows that end-point titration and duplex PCR can be used as a simple and rapid method to quantitate CMV DNA in blood of patients that are either CMV-positive or CMV-negative by cell culture. Quantitation of CMV DNA in blood by end-point titration PCR has potential to differentiate between asymptomatic CMV infection and symptomatic CMV disease, and to monitor viral load during viral therapy.