Format

Send to:

Choose Destination
See comment in PubMed Commons below
J Virol Methods. 2000 Jan;84(1):1-14.

Detection and quantitation of human cytomegalovirus DNA in faeces.

Author information

  • 1Department of Virology, Academic Medical Center, University of Amsterdam, The Netherlands. w.r.boom@amc.uva.nl

Abstract

The development and performance of a robust and sensitive PCR assay are described for the detection and quantitation of human cytomegalovirus DNA in human faecal specimens. In this assay, CMV DNA was purified by an optimised DNA extraction protocol together with internal control DNA that monitored both DNA extraction efficiency and PCR efficiency. The lower detection limit of the assay was reached at about 100 CMV particles per ml of (25-50%) faecal suspension. CMV DNA could be quantitated in the range of about 300-100000 molecules per ml of faecal suspension. CMV DNA loads obtained in clinical faeces specimens suggest that the assay can be used to monitor the efficacy of antiviral treatment. Reconstruction experiments that monitored the efficiency of DNA extraction of a preliminary DNA extraction protocol, showed low DNA yields for 9% of the specimens (n = 78). In all cases, low DNA extraction efficiency seemed to be due to a component present in faeces that prevented DNA binding to silica particles, presumably by competitive binding. Choosing the right ratio of silica particles to faeces specimen solved this problem. Similarly, reconstruction experiments showed that the strong PCR inhibition that was observed in 8% of the specimens could effectively be relieved by the inclusion of alpha-casein in the PCR mixtures.

PMID:
10644082
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Write to the Help Desk