Fedele C G, Ciardi M, Delia S, Echevarria J M, Tenorio A
Diagnostic Microbiology Service, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain.
J Virol Methods. 1999 Oct;82(2):137-44. doi: 10.1016/s0166-0934(99)00095-6.
A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.
开发了一种新型多重巢式PCR（nPCR）方法，用于在单管中同时检测和区分多瘤病毒JC、BK和SV40的DNA。在第一轮扩增步骤中，使用同一组引物扩增JCV、BKV和SV40大T抗原基因的保守DNA区域。第二轮多重nPCR使用一组引物进行，这些引物设计用于为每种相关病毒生成不同大小的产物。系统优化热循环参数和每个反应组分的浓度，以实现nPCR检测的最佳特异性和灵敏度。该方法的灵敏度在多瘤病毒基因组的1至10个拷贝之间。对有进行性多灶性白质脑病（PML）临床和神经放射学证据的艾滋病患者的脑脊液（CSF）以及有其他神经改变的艾滋病患者的CSF进行了检测。还对受出血性膀胱炎影响的骨髓移植受者的尿液标本进行了检测。获得的结果表明，该检测方法是支持多瘤病毒感染诊断的良好工具，可用于流行病学目的以及其他研究，以便更好地确定多瘤病毒在人类疾病中的作用。
