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Journal of Clinical Microbiology, July 2002, p. 2381-2386, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2381-2386.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Jason L. Sanchez1,{dagger} and Gregory A. Storch1,2*

Departments of Medicine,1 Pediatrics, Washington University School of Medicine, St. Louis, Missouri 631102

Received 19 November 2001/ Returned for modification 28 February 2002/ Accepted 13 April 2002

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 106 human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 101 to 106 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.

* Corresponding author. Mailing address: Department of Pediatrics, Campus Box 8116, Washington University School of Medicine at St. Louis Children's Hospital, 1 Children's Pl., Room 2N-68, St. Louis, MO 63110. Phone: (314) 454-6079. Fax: (314) 454-2274. E-mail: storch{at}kids.wustl.edu.

{dagger} Present address: Infectious Disease Consultants, Wayzata, MN 55446.

Journal of Clinical Microbiology, July 2002, p. 2381-2386, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2381-2386.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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