Improved amplification of cytomegalovirus DNA from urine after purification of DNA with glass beads.
Cytomegalovirus can be detected in a variety of specimens including leukocytes, urine, saliva, feces, and various tissues by polymerase chain reaction (PCR) amplification of viral DNA. Although methods for amplification are fairly standard, sample preparation is not well characterized, especially for tissue. Typically, preparation of samples for PCR amplification ranges from simple boiling to phenol/chloroform extraction and quantification before testing. Several reports have described inhibition of the PCR in some samples types. Here we show that reliable detection of cytomegalovirus DNA in urine is obtained only after some degree of DNA purification, presumably because of PCR inhibition by a yet unidentified component present in a few of the urine samples tested. Glass, in the form of fine beads, was used to adsorb DNA such that protein and other substances could be selectively eluted before the recovery of DNA for PCR amplification. Urine samples prepared by this method did not show inhibition, and results correlated well with those by tissue culture for detection of cytomegalovirus.
Buffone
G J
GJ
Department of Pathology, Baylor College of Medicine, Houston, TX 77030.
Demmler
G J
GJ
Schimbor
C M
CM
Greer
J
J
D000163:Acquired Immunodeficiency Syndrome / Q000150:complications; D000328:Adult; D003587:Cytomegalovirus / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000150:complications / Q000382:microbiology* / Q000652:urine; D004279:DNA, Viral / Q000235:genetics / Q000652:urine*; D005188:False Negative Reactions; D005898:Glass; D006358:Hot Temperature; D006801:Humans; D008863:Microspheres; D016133:Polymerase Chain Reaction
D004279:DNA, Viral