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Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell‐free cytomegalovirus
Author(s) -
Tedder R.S.,
Ayliffe U.,
Preiser W.,
Brink N.S.,
Grant P.R.,
Peggs K.S.,
Mackin S.,
KreigSchneider F.,
Kirk S.,
Garson J.A.
Publication year - 2002
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.2175
Subject(s) - human cytomegalovirus , virology , biotinylation , biology , polymerase chain reaction , cytomegalovirus , real time polymerase chain reaction , microbiology and biotechnology , concordance , betaherpesvirinae , herpesviridae , virus , viral disease , bioinformatics , genetics , gene
Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and “in‐house” assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV‐specific and MCMV‐specific enzyme‐labelled probes and automated chemiluminescence detection. Log‐transformed HCMV‐to‐MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV‐spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be ≥ 4 log 10 . A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity. J. Med. Virol. 66:518–523, 2002. © 2002 Wiley‐Liss, Inc.