Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase.
A 24 base pair oligonucleotide probe directly conjugated to alkaline phosphatase has been used to detect immobilised amplicons derived from a cytomegalovirus specific polymerase chain reaction (PCR). The sensitivity of detection using a highly amplified alkaline phosphatase detection system was four genome equivalents and was comparable to the limit 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 from four genome equivalents (absorption at 492 nm: 0.132). The assay was used to identify the presence of cytomegalovirus in blood DNA extracts from immunocompromised patients in whom conventional ethidium bromide stained agarose gel electrophoresis revealed the presence of multiple amplicons. Samples yielding an uninterpretable result at both neat and diluted 1 in 20 in the PCR gave rise to the highest proportion of positive results (68%) whilst samples that produced uninterpretable results neat but were negative at 1 in 20 and vice versa gave positive rates of 33.6 and 21.7%, respectively. The use of this assay for identifying cytomegalovirus specific PCR products in problematic samples is discussed.
Gor
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Department of Virology, Royal Free Hospital, Hampstead, London, UK.
Lee
D
D
Emery
V C
VC
D000469:Alkaline Phosphatase / Q000737:chemistry*; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D000469:Alkaline Phosphatase