Monitoring human cytomegalovirus infection with nested PCR: comparison of positive rates in plasma and leukocytes and with quantitative PCR

Abstract

BackgroundHuman cytomegalovirus (HCMV) infection poses a significant health threat to immunocompromised individuals. Here we performed this study to set up a highly sensitive nested PCR method applicable for detecting HCMV infection in high-risk individuals. In this work, 106 blood specimens from 66 patients with potential HCMV infection were obtained. Total DNA was extracted separately from plasma and peripheral blood leukocytes (PBL) of each sample. HCMV DNA was detected in parallel by nested PCR and quantitative real-time PCR (qRT-PCR), and the results were compared.ResultsSerial dilution test revealed that the detection limit of nested PCR was 180 copies/ml. The nested PCR showed a higher positive rate than qRT-PCR (34.9% vs. 12.3%, p < 0.001). The positive rate of nested PCR based on PBL DNA was significantly higher than that based on plasma DNA (34.9% vs. 18.9%, p = 0.002). Of the 14 patients with serial samples, 11 were positive for HCMV DNA in PBL while only 7 were positive in plasma. Moreover, for each patient, nested PCR using PBL DNA also detected more positive samples than that using plasma DNA.ConclusionCombined use of nested PCR with PBL DNA is highly sensitive in defining HCMV infection. This assay is particularly useful in the case of quantification not essential.