Gor D, Lee D, Emery V C
Department of Virology, Royal Free Hospital, Hampstead, London, UK.
J Virol Methods. 1996 Sep;61(1-2):145-50. doi: 10.1016/0166-0934(96)02081-2.
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.
一种直接与碱性磷酸酶偶联的24碱基对寡核苷酸探针已被用于检测源自巨细胞病毒特异性聚合酶链反应（PCR）的固定化扩增子。使用高度扩增的碱性磷酸酶检测系统的检测灵敏度为四个基因组当量，与使用琼脂糖凝胶方法的检测限相当。已知不含巨细胞病毒DNA的样品在492nm处的平均光密度为0.085±0.006，与四个基因组当量产生的光密度（492nm处的吸光度：0.132）有很好的区分。该检测方法用于鉴定免疫功能低下患者血液DNA提取物中巨细胞病毒的存在，在这些患者中，传统的溴化乙锭染色琼脂糖凝胶电泳显示存在多个扩增子。在PCR中 neat 和 1:20稀释时均产生无法解释结果的样品产生阳性结果的比例最高（68%），而 neat 时产生无法解释结果但1:20稀释时为阴性的样品以及反之亦然的样品的阳性率分别为33.6%和21.7%。讨论了该检测方法在有问题的样品中鉴定巨细胞病毒特异性PCR产物的用途。
