Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA

Abstract

We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 10 6 human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 10 1 to 10 6 copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.Cytomegalovirus (CMV) continues to be an important cause of morbidity and occasional mortality in immunocompromised patients. While the impact of CMV has been lessened by the use of ganciclovir, concerns including drug toxicity, cost, and drug resistance have stimulated efforts to find the most efficient strategies for using the drug, some of which involve using a diagnostic test to monitor for infection (15). The ideal diagnostic test should be sufficiently sensitive to detect infection at an early stage before clinically significant disease has occurred, but not so sensitive that only a small proportion of patients with a positive test would actually develop disease. Quantitative nucleic acid detection assays are the most likely to be useful in this way because they combine inherent analytic sensitivity with the ability to define threshold levels that could be used to initiate treatment. A number of studies have shown relationships between the level of CMV DNA in blood and the likelihood of disease (2, 5, 7, 10-13, 17, 20, 26). The stability of DNA is an additional advantage that is important because specimens may have to be transported before testing (18, 21).The development of real-time PCR technology has simplified nucleic acid quantification and is coming into more widespread use. Real-time, quantitative PCR has several advantages over older forms of quantitative PCR assays. Experience to date has reported comparable sensitivity but superior reproducibility and precision compared to previous methods, with a wide dynamic range (14, 19). The fluorescence-based real-time assays also allow for multiple...

Once daily ganciclovir as initial pre-emptive therapy delayed until threshold CMV load ⩾10000 copies/ml: a safe and effective strategy for allogeneic stem cell transplant patients

“…16 Inhomogeneity of leukocyte lysates can lead to variability and inaccuracies in results. Inconsistencies in procedures for storage of blood samples and separation of plasma from whole blood can lead to artificially increased CMV viral loads secondary to lysis of cells.…”

“…Quantitative CMV PCR was performed as described previously. 16 Briefly, whole-blood samples were processed by the MagNa Pure automated extractor using the MagNa Pure LC Total Nucleic Acid Isolation Kit. The LightCycler real-time PCR instrument was used to amplify, detect, and quantify CMV DNA from whole-blood samples, using a 5 0 exonuclease ('Taqman') assay.…”

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 interest of multiplex, quantitative, and real-time PCR assays that makes it possible to normalize viral load according to the amount of cellular DNA has been underlined previously in a number of studies. In the setting of CMV or EBV infections, such methods have first been developed on separated leukocytes [Jabs et al, 2001], and more recently on whole blood [Sanchez and Storch, 2002].…”

“…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).…”

Assessment of the Human Cytomegalovirus UL97 Gene to identify the Resistance to Ganciclovir in Kidney Transplant Recipients in Labbafi-Nejad Hospital

“…Human cytomegalovirus (HCMV) as one of the common virus, is epidemiologically varies in different regions of the world and between socioeconomic and age groups (1,2). Therefore, for the accurate treatment of HCMV, diagnosis in an early stage is especially necessary to avoid poor clinical outcomes (3)(4)(5). Ganciclovir is a nucleoside analogue antiviral drug which is widely used to treat systemic CMV disease.…”