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首页> 外文期刊>Journal of Virological Methods >Optimisation of the polymerase chain reaction and dot-blot hybridisation for detecting cytomegalovirus DNA in urine: comparison with detection of early antigen fluorescent foci and culture.
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Optimisation of the polymerase chain reaction and dot-blot hybridisation for detecting cytomegalovirus DNA in urine: comparison with detection of early antigen fluorescent foci and culture.

机译：用于检测尿液中巨细胞病毒DNA的聚合酶链反应和点印迹杂交的优化：与早期抗原荧光灶和培养物的检测比较。

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Rapid, sensitive and specific assays are required for the diagnosis of CMV infection following transplantation. We describe our experience in developing assays for detecting CMV in urine. Conventional preparation of probes cloned after amplification in E. coli led to contamination with E. coli nucleic acids; these hybridised to E. coli DNA present in urine and produced false positive results. Two CMV probes (Hind III and gL) hybridised to human DNA despite high stringency; these probes were thus unsuitable for detecting viral nucleic acids in clinical samples. A PCR derived probe from the immediate early gene of CMV detected dot-blotted CMV DNA specifically. Optimal preparation of urine for detection of CMV DNA was as follows; four freeze/thaw cycles and ultracentrifugation before in vitro proteinase K/SDS treatment, phenol:chloroform extraction, heat denaturation and direct application onto a nylon membrane. However, dot-blot hybridisation was a poor test for CMV in urine; it had low sensitivity and specificity compared with virus isolation and DEAFF. Single round PCR of a 293 bp region of CMV DNA was sensitive and specific to CMV targets. However, undiluted urine contained PCR inhibitors that could only be partly removed by using PEG precipitation. PCR of CMV DNA from urine was specific but was insensitive compared to conventional culture and DEAFF. A significant proportion of urine samples were toxic in conventional culture and DEAFF tests but, PCR of CMV DNA from urine is insensitive and despite its specificity is unlikely to be advantageous in clinical practice even when DEAFF or culture prove unreliable.
机译：诊断移植后CMV感染需要快速，灵敏和特异的检测方法。我们描述了我们在开发检测尿液中巨细胞病毒的检测方法方面的经验。常规的在大肠杆菌中扩增后克隆的探针的制备导致大肠杆菌核酸的污染。它们与尿液中存在的大肠杆菌DNA杂交并产生假阳性结果。尽管高度严格，但仍有两个CMV探针（Hind III和gL）与人DNA杂交;因此，这些探针不适用于检测临床样品中的病毒核酸。 PCR衍生自CMV的早期早期基因的探针可特异性检测斑点印迹的CMV DNA。用于检测CMV DNA的最佳尿液制备如下：在体外蛋白酶K / SDS处理，苯酚：氯仿提取，热变性和直接应用于尼龙膜上之前，先进行四个冷冻/解冻循环并进行超速离心。然而，斑点杂交技术不能很好地检测尿液中的巨细胞病毒。与病毒分离和DEAFF相比，它的敏感性和特异性低。 CMV DNA 293 bp区域的单轮PCR对CMV靶标敏感且特异。但是，未稀释的尿液中含有PCR抑制剂，只能通过使用PEG沉淀部分除去。尿液中CMV DNA的PCR是特异性的，但与常规培养物和DEAFF相比不敏感。在常规培养和DEAFF测试中，很大一部分尿液样品是有毒的，但是，从尿液中提取CMV DNA并不敏感，尽管其特异性即使在DEAFF或培养物证明不可靠的情况下也不太可能在临床实践中发挥作用。

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