Kiderlen Albrecht F, Radam Elke, Lewin Astrid
Robert Koch Institute, Cellular Immunology Unit P22, Nordufer 20, 13353 Berlin, Germany.
BMC Microbiol. 2008 Dec 3;8:210. doi: 10.1186/1471-2180-8-210.
The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in - apparently - immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research.
A real-time polymerase chain reaction assay using TaqMan probes (real-time PCR) was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5) genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species), nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG), human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results.
A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.
自由生活的阿米巴原虫曼氏巴贝斯虫可在免疫功能低下以及看似免疫功能正常的人类和其他哺乳动物物种中引发致命性脑炎。快速、特异、灵敏且可靠的检测方法几乎不需要病原体特异性专业知识，这是成功治疗的绝对前提，也是实验研究和流行病学研究的理想工具。
建立了一种使用TaqMan探针的实时聚合酶链反应检测方法（实时PCR），专门针对曼氏巴贝斯虫的核糖核酸酶P基因。该检测方法可检测到在无菌培养中生长的至少2个（低至0.5个）曼氏巴贝斯虫基因组。它与密切相关的棘阿米巴属（3个物种）的DNA无反应，也与刚地弓形虫、硕大利什曼原虫、鼠肺孢子菌、牛分枝杆菌（卡介苗）、人脑、各种小鼠器官或人类和小鼠细胞系的DNA无反应。该检测方法能有效检测加样细胞培养物、加样小鼠器官匀浆、感染曼氏巴贝斯虫的小鼠以及2例经证实的脑巴贝斯虫病患者的中枢神经系统组织DNA制剂中的曼氏巴贝斯虫DNA。这套新型引物成功地与一套已发表的针对曼氏巴贝斯虫18S rRNA基因的引物组合，用于双重实时PCR检测，以确保最大特异性，并预防假阴性结果。
本文介绍了一种针对曼氏巴贝斯虫的实时PCR检测方法，该方法高度特异、灵敏且可靠，因此适用于诊断和研究。
