Detection and quantitation of human cytomegalovirus DNA in faeces.
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.
Boom
R
R
Department of Virology, Academic Medical Center, University of Amsterdam, The Netherlands. w.r.boom@amc.uva.nl
Sol
C
C
Weel
J
J
Lettinga
K
K
Gerrits
Y
Y
van Breda
A
A
Wertheim-Van Dillen
P
P
D000328:Adult; D001483:Base Sequence; D002364:Caseins; D003093:Colitis, Ulcerative / Q000175:diagnosis / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D005243:Feces / Q000821:virology*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D002364:Caseins; D017931:DNA Primers; D004279:DNA, Viral; D012822:Silicon Dioxide