Detection and Identification of Microsporidia in Stool

粪便中微孢子虫的检测与鉴定

• 批准号: 6103681
• 负责人: DANIEL P. FEDORKO
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6103681
• 关键词: Microsporidia; communicable disease diagnosis; diagnosis design /evaluation; feces analysis; gastrointestinal infection; microorganism culture; microorganism genetics; nucleic acid probes; polymerase chain reaction; rapid diagnosis; ribosomal RNA; species difference

Several reports have appeared recently in the literature detailing cases in which gastrointestinal disease, often with significant associated morbidity, occurred in acquired immune deficiency syndrome patients infected with microsporidial parasites. As a consequence of these reports, there has been an increased awareness among clinicians of the potential importance of microsporidia as gastrointestinal (GI) pathogens. Attention is now being focused on methods for both diagnosis and treatment. There is considerable interest in determining the diagnostic value of stool specimens for detecting GI disease due to microsporidia. A variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is clearly required. We have published a report that describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. The PCR primers used in the assay will amplify all of the currently identified microsporidial human pathogens. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project will be to determine the sensitivity of our assay and the length of time specimens can be stored in formalin and still allow detection of the microsporidia. We have developed a shorter, cheaper, and less rigorous method for extraction of microsporidial DNA from stool.

最近有几份报告出现在 文献详细描述了胃肠道疾病的病例， 与显著相关的发病率，发生在获得性免疫 感染微孢子虫的虚证患者。 由于这些报告， 临床医生对以下潜在重要性的认识 微孢子虫是胃肠道（GI）病原体。现在注意力 专注于诊断和治疗的方法。那里 在确定粪便的诊断价值方面， 用于检测微孢子虫引起的胃肠道疾病的标本。各种 的染色模式进行了评价， 在粪便制备物中观察微孢子虫。可惜 感染人类的各种微孢子虫， 只有通过观察特征性的亚细胞 组织电子显微照片中生物体的形态特征 标本因为目前治疗方案的有效性 发展可能是某种特定的物种，因为 为了获得合适的标本， 对于电子显微镜（EM），一种更广泛使用的方法， 比EM识别是明确需要的。我们发布了一个 该报告描述了我们的聚合酶链反应（PCR）检测， 微孢子虫rRNA基因扩增后进行限制性内切酶切 PCR产物的核酸内切酶消化作为鉴定 新鲜粪便中有微孢子虫。检测中使用的PCR引物将 扩增所有目前鉴定的人类微孢子虫 病原体我们正在继续修改检测方法， 检测福尔马林固定粪便标本中的微孢子虫。一 使用PCR扩增的DNA的Southern印迹格式和 已经开发了种特异性DNA探针用于 微孢子虫物种形成该项目的最后阶段将是 确定我们检测的灵敏度和时间长度 标本可以保存在福尔马林中， 微孢子虫我们已经开发出一种更短，更便宜， 从粪便中提取微孢子虫DNA的严格方法。