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.

FIG. 1.

Relationship between the C_T and log₁₀ CMV copy number, showing linearity. The CMV copy number represents copies of plasmid pJSCPOL.

FIG. 2.

Serial dilutions of a CMV-positive patient specimen, demonstrating the linearity of both the CMV and human DNA PCR assays. The calculated CMV viral load (CMV copies per microgram of human DNA) remained stable despite dilution of the original specimen.

FIG. 3.

Effects of interval from blood draw to plasma separation and storage temperature on levels of human DNA in plasma. Panels A and B show data for two different volunteers.

FIG. 4.

Effect of plasma separation method on the amount of human DNA in plasma. The human DNA copy number was measured in specimens from four different volunteers after each specimen was processed according to each of four different procedures.