Polymerase chain reaction assay for detection of human cytomegalovirus

Abstract

Direct detection of human cytomegalovirus (HCMV) from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying HCMV DNA. The efficiency of the amplification reaction was examined by using three different buffers and concentrations of deoxynucleotide triphosphates. The PCR assay was most efficient with a reaction mixture containing 17 mM ammonium sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each deoxynucleotide triphosphate at a final concentration of 1.5 mM. After 35 cycles of amplification, 0.15 fg of a plasmid containing the cloned target gene (corresponding to approximately six gene copies) was detected. The PCR assay correctly identified all of 24 clinical isolates of HCMV. Virus in urine specimens could be disrupted by heating at 93 degrees C for 30 min. The viral DNA was amplified directly from 5 microliters of preheated urine, with no further treatment before amplification. We tested the PCR assay on urine specimens from patients who had undergone renal transplantation that had been screened for the presence of HCMV by enzyme-linked immunosorbent assay, hybridization assay, and direct virus isolation. Specimens that were positive by one or more of these assays were screened by PCR. HCMV was consistently detected by PCR in all specimens that were positive by at least one other test. No cross-reactivity to other herpesviruses or MRC-5 cellular DNA was observed.