Quantification of Human Cytomegalovirus DNA by Real-Time PCR

Abstract

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).Human cytomegalovirus (HCMV) infection is characterized by a primary infection leading to a lifelong persistence of the viral genome. Periodically, the virus reactivates from latency and recovers its ability to multiply. HCMV is a major cause of morbidity and mortality in bone marrow or solid-organ transplant recipients and in AIDS patients. Early diagnosis of HCMV infection in high-risk patients is essential in order to start preemptive treatments (3,13,15). The detection of the pp65 antigen in leukocytes is a sensitive method widely used for the early diagnosis of HCMV infection, but it is laborintensive, requires immediate processing, and relies on a subjective interpretation of the slides (1, 7). Qualitative PCR detection of HCMV DNA in leukocytes or plasma is considered the most sensitive method, but it lacks specificity for the diagnosis of HCMV disease (2, 6, 16). Quantification of HCMV DNA has been proposed to be more specifically associated with the disease (1, 18). Real-time PCR based on the TaqMan technology (4, 5) provides an accurate means to quantify viral DNA with the major advantage of avoiding post-PCR handling that can be the source of DNA carryover. Recent studies report the utility of real-time PCR for the quantification of HCMV DNA (9,11,12,14,17). In these studies, the primers used for PCR were located in the major immediate-early gene (12,14,17), the US17 gene (9), or the glycoprotein B gene (17). As suggested by Yun et al., the sensitivity of quantitative PCR may be dependent on the viral target gene (17); however, the most appropriate region for amplification has not been established, as the sequence diversity of clinical isolates remains to be characterized. In the present study, a real-time PCR assay was developed to quantify HCMV DNA in peripheral blood leukocytes (PBLs) using a target sequence located in the UL83 gene which codes for the lower matrix protein detected in the pp65 antigenemia test. The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin) and the results were compared to those of the pp65 antigenemia assay.DNA extractions were performed in all experiments using the QIAamp blood kit (QIAGEN S. A., Courtaboeuf, France) according to the manufacturer's recommendations, except that DNA was eluted in 200 l of distilled water. To amplify HCMV DNA, primers and probe were defined in the UL83 region as follows: pp549s (direct primer), 5Ј-GTCAGCGTTC GTGTTTCCCA-3Ј; pp812as (reverse primer), 5Ј-GGGACAC AACACCGTAAAGC-3Ј; and pp770s (fluorogenic probe), 5ЈFAM-CCCGCAACCCGCAACCCTTCATG-3ЈTAMRA. No cross-reactivity was observed when the specificity of the primers and probe was tested for other human herp...

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation

“…[12][13][14][18][19][20][21] However, they utilized different methods including regions for primers and TaqMan probe, and the source of DNA extraction (plasma, whole blood or leukocyte). Therefore, we must carefully interpret the results of real-time PCR.…”

Combined chemotherapy including high‐dose methotrexate in KSHV/HHV8‐associated primary effusion lymphoma

“…Standard curves were obtained with serial 10-fold dilutions of viral stock prepared with Namalwa cell line harboring two EBV copies per cell [9]. EBV standard curves have previously been characterized and normalized using the standard curve from human albumin gene and specific sets of primers and 5Ј FAM labeled-probes sequence [9,10]. Amplifications conditions have previously been described [7].…”

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

“…The efficacy of extraction and amplification was evaluated by comparing the calculated number of cells after albumin PCR with blood cell counts on the same sample. The overall reaction efficiency (median 62%) is satisfying regarding the few other published studies that evaluated manual or automated extraction efficiency [Gault et al, 2001;Gouarin et al, 2004]. Correlation between the number of cells calculated by quantitative albumin PCR and blood cell count is much better for samples <5.0 Â 10 9 PBLs/ ml.…”

“…The monitoring of the efficiency of antiviral therapy is based on viral DNA load intensity and kinetics and requires accurate and reproducible monitoring. Most clinical centers have developed in house molecular assays, based on real-time PCR technology that offer high performance and can be completed within 1 day [Niesters et al, 2000;Gault et al, 2001;Griscelli et al, 2001;Sanchez and Storch, 2002;Boeckh et al, 2004;FafiKremer et al, 2004;Niesters, 2004;Piiparinen et al, 2004]. There are many differences between these methods, due to different clinical samples (whole blood, peripheral blood leucocytes [PBL], and plasma), manual or automated extraction procedures, and PCR methods (real-time PCR performances, use of internal control).…”