Franzen C, Müller A, Hartmann P, Hegener P, Schrappe M, Diehl V, Fätkenheuer G, Salzberger B
Department of Internal Medicine I, University of Cologne, Germany.
Folia Parasitol (Praha). 1998;45(2):140-8.
Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E. intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSBlue competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.
聚合酶链反应（PCR）技术已被开发用于检测不同生物样本中的微孢子虫DNA。我们通过PCR、Southern印迹杂交、限制性内切酶消化分析、克隆和比较基因测序，利用rRNA基因的序列数据，在HIV感染患者的不同生物样本中鉴定微小隐孢子虫、肠脑炎微孢子虫、兔脑炎微孢子虫和海伦脑炎微孢子虫。使用一对引物从电子显微镜确认感染的样本中扩增微小隐孢子虫、肠脑炎微孢子虫和海伦脑炎微孢子虫的整个小亚基（SSU）-rRNA基因。扩增得到的1.2 kb SSU-rRNA基因片段被连接到pMOSBlue T载体中，转染到pMOSBlue感受态细胞中，并用作阳性对照。使用几对引物和杂交探针从不同样本中扩增和鉴定微孢子虫DNA。对所有患者的样本进行了光学显微镜检查，并对一部分患者样本进行了透射电子显微镜检查。从所有确诊有微孢子虫感染的样本中获得了DNA产物。通过Southern印迹杂交、限制性内切酶消化分析或DNA测序确定DNA片段的身份。结果表明，PCR是HIV感染患者不同生物样本中微孢子虫DNA存在的可靠且灵敏的指标。PCR可进一步用于微孢子虫的种属鉴别，甚至可用于鉴别光镜和/或电镜无法区分的种属。
