Detection and quantitation of human cytomegalovirus DNA in faeces

The development and performance of a robust and sensitive PCR assay are des cribed for the detection and quantitation of human cytomegalovirus DNA in h uman faecal specimens. In this assay, CMV DNA was purified by an optimised DNA extraction protocol together with internal control DNA that monitored b oth DNA extraction efficiency and PCR efficiency. The lower detection limit of the assay was reached at about 100 CMV particles per mi of (25-50%) fae cal suspension. CMV DNA could be quantitated in the range of about 300-100 000 molecules per mi of faecal suspension. CMV DNA loads obtained in clinic al faeces specimens suggest that the assay can be used to monitor the effic acy of antiviral treatment. Reconstruction experiments that monitored the e fficiency of DNA extraction of a preliminary DNA extraction protocol, showe d low DNA yields for 9% of the specimens (n = 78). In all cases, low DNA ex traction efficiency seemed to be due to a component present in faeces that prevented DNA binding to silica particles, presumably by competitive bindin g. Choosing the right ratio of silica particles to Faeces specimen solved t his problem. Similarly, reconstruction experiments showed that the strong P CR inhibition that was observed in 8% of the specimens could effectively be relieved by the inclusion of alpha-casein in the PCR mixtures. (C) 2000 El sevier Science B.V. All rights reserved.