Quantitation of human cytomegalovirus DNA in plasma using an affordable in-house qPCR assay

Quantitation of human cytomegalovirus (HCMV) DNA is used to monitor immunocompromised patients in order to identify patients for preemptive therapy. Although several commercial qPCR assays are available for quantitation of HCMV, their major disadvantage is the high cost. In the present study, an internally controlled quantitative real-time PCR assay based on hydrolysis probe technology was developed for detection and quantitation of HCMV DNA in plasma samples. To demonstrate its performance characteristics, a total of 178 plasma samples from 102 kidney and hematopoietic stem cell transplanted patients were tested. The assay showed good precision and reproducibility, and an analytical sensitivity of 288.5 copies/ml or 17.6 copies/reaction. A sensitivity of 93.1% and a specificity of 96.6% were determined by examining clinical samples. Analysis of a panel containing potentially interfering viruses demonstrated no cross-reactivity with the assay. A strong correlation was observed between this qPCR method and the commercial Artus® CMV RG PCR kit (R = 0.948; P = 0.000). These results indicate that the affordable internally controlled qPCR method described will be useful for monitoring HCMV infection in plasma samples of immunocompromised patients.

► A qPCR assay was developed for use as a low-cost approach for CMV quantitation.
► Designed primer-probe set considers all available strains of CMV that have been reported in National Center for Biotechnology Information (NCBI).
► The assay was validated accurately to fulfill the minimum information for publication of quantitative real-time PCR experiments (MIQE) requirements.
► The in-house assay has a good correlation with a reliable commercial kit.