title: The development of a quantitative PCR ELISA to determine HCMV DNAaemia levels in heart, heart/lung and lung transplant recipients.
abstract: The adaptation of the polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) using a co-amplified DNA standard to quantitate the human cytomegalovirus (HCMV) glycoprotein B (gB) gene in clinical samples is described. The PCR ELISA is a solution hybridisation system with colorimetric end stage detection of amplicons. A DNA internal standard (IS) was designed by replacing a probe sequence used currently within the gB region with a heterogeneous sequence, allowing co-amplification with the same oligonucleotide primer sets and differentiation by probe hybridisation. Two DNA fragments homologous to the gB and IS sequences were generated and used for co-amplification studies to construct a standard curve. From this the copy number of the gB gene present in clinical samples could be interpolated. Co-amplification with 1000 IS copies allowed quantitation of 10-1000000 gB DNA copies in a single PCR. This assay was validated subsequently using blood samples tested by the HCMV antigenaemia assay and showed a trend of increasing HCMV DNAaemia with increasing antigenaemia levels. This rapid assay avoids using gel electrophoresis and cumbersome quantitative systems. It has the potential for early identification of patients at high risk of developing HCMV disease, and for therapeutic monitoring.
authors.affiliation: Public Health Laboratory, Withington Hospital, Manchester, UK. lisa.barber@man.ac.uk
authors.forename: L
authors.initials: L
authors.lastname: Barber
authors.forename: J J
authors.initials: JJ
authors.lastname: Egan
authors.forename: A J
authors.initials: AJ
authors.lastname: Turner
authors.forename: M
authors.initials: M
authors.lastname: Guiver
authors.forename: A A
authors.initials: AA
authors.lastname: Woodcock
authors.forename: N
authors.initials: N
authors.lastname: Yonan
authors.forename: A K
authors.initials: AK
authors.lastname: Deiraniya
authors.forename: A J
authors.initials: AJ
authors.lastname: Fox
mesh_terms: D001483:Base Sequence; D002138:Calibration; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000821:virology*; D004279:DNA, Viral / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016027:Heart Transplantation; D016041:Heart-Lung Transplantation; D006801:Humans; D016040:Lung Transplantation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011183:Postoperative Complications / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics*; D014766:Viremia / Q000175:diagnosis
chemical_list: D004279:DNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus