DETECTION OF CYTOMEGALOVIRAL DNA IN HUMAN-MILK CELLS AND CELL-FREE MILK WHEY BY NESTED PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartm ents of human milk. A protocol for the preparation of milk whey free o f fat and cells for the detection of human cytomegalovirus (HCMV) by n ested PCR is presented. This is based upon the experience of the separ ation of more than 200 milk specimens of healthy seropositive breast f eeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. I n limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectab le by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depende d on a minimum number of milk cells (10(5)-2 x 10(5)) available for DN A extraction. In contrast to the findings of cytomegaloviral DNA in na tive sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an in hibition of Tag polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequ ential milk specimens, in some cases the presence of HCMV DNA in colos trum could be demonstrated. DNAlactia of milk cells and whey was parti ally discordant. Onset (week 1-4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual pat terns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropos itive mothers. (C) 1998 Elsevier Science B.V. All rights reserved.