J Virol Methods. 1994 Sep;49(2):195-208. Unique Identifier : AIDSLINE
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 microgram 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.
Adult AIDS-Related Opportunistic Infections/BLOOD/*DIAGNOSIS Base
Sequence Blotting, Southern/METHODS Cloning, Molecular Comparative
Study Cytomegalovirus/GENETICS/*ISOLATION & PURIF Cytomegalovirus
Infections/BLOOD/*DIAGNOSIS DNA/ANALYSIS DNA, Viral/ANALYSIS/*BLOOD
Escherichia coli Female Human Kidney Transplantation
Leukocytes/*VIROLOGY Male Middle Age Molecular Sequence Data
Oligonucleotide Probes Placenta Plasmids Polymerase Chain
Reaction/*METHODS Pregnancy Reproducibility of Results Restriction
Mapping CLINICAL TRIAL JOURNAL ARTICLE