DETECTION OF HUMAN CYTOMEGALOVIRUS POLYMERASE CHAIN-REACTION PRODUCTSUSING OLIGONUCLEOTIDE PROBES DIRECTLY CONJUGATED TO ALKALINE-PHOSPHATASE

A 24 base pair oligonucleotide probe directly conjugated to alkaline p hosphatase has been used to detect immobilised amplicons derived from a cytomegalovirus specific polymerase chain reaction (PCR). The sensit ivity of detection using a highly amplified alkaline phosphatase detec tion system was four genome equivalents and was comparable to the limi t of detection using agarose gel methods. The mean optical density at 492 nm of samples not known to contain cytomegalovirus DNA was 0.085 /- 0.006 and was well separated from the optical density generated fro m four genome equivalents (absorption at 492 nm: 0.132). The assay was used to identify the presence of cytomegalovirus in blood DNA extract s from immunocompromised patients in whom conventional ethidium bromid e stained agarose gel electrophoresis revealed the presence of multipl e amplicons. Samples yielding an uninterpretable result at both neat a nd diluted 1 in 20 in the PCR gave rise to the highest proportion of p ositive results (68%) whilst samples that produced uninterpretable res ults neat but were negative at 1 in 20 and vice versa gave positive ra tes of 33.6 and 21.7%, respectively. The use of this assay for identif ying cytomegalovirus specific PCR products in problematic samples is d iscussed.