Detection and Identification of Microsporidia in Stool

粪便中微孢子虫的检测与鉴定

• 批准号: 6431839
• 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/6431839
• 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

There is considerable interest in determining the diagnostic value of stool specimens for detecting gastrointestinal disease caused by Microsporidia organisms, and 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 required. We have published a report which 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. 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 has been to determine the sensitivity of our assay and the length of time the specimens can be stored in formalin and still allow detection of the microsporidia. We have demonstrated that prolonged storage of microsporidia in formalin or polyvinyl alcohol reduces the chances of amplification by PCR. We suggest washing stool specimens after fixation in formalin and storage of the washed specimen in phosphate-buffered saline. We have developed a shorter, cheaper, and simpler method for extraction of microsporidial DNA from stool.

有相当大的兴趣，在确定粪便标本的诊断价值，用于检测微孢子虫生物体引起的胃肠道疾病，和各种染色方式已被评估其可视化微孢子虫在粪便制剂的能力。不幸的是，感染人类的各种微孢子虫只能通过在组织标本的电子显微镜照片中观察生物体的特征性亚细胞形态特征来准确识别。由于目前正在开发的治疗方案的疗效可能有些种属特异性，因为侵入性程序是必要的，以获得适当的标本进行电子显微镜（EM），一个更广泛可用的方法来识别比EM是必要的。我们已经发表了一份报告，其中描述了我们的聚合酶链反应（PCR）检测微孢子虫rRNA基因的扩增，然后用限制性内切酶消化PCR产物作为一种方法来识别新鲜粪便中的微孢子虫。我们正在继续改进检测方法，以检测福尔马林固定粪便标本中的微孢子虫。使用PCR扩增的DNA和种特异性DNA探针的Southern印迹格式已被开发用于微孢子虫物种形成。该项目的最后阶段是确定我们的检测方法的灵敏度和样本可以在福尔马林中储存的时间长度，并且仍然允许检测微孢子虫。我们已经证明，长期储存的微孢子虫在福尔马林或聚乙烯醇减少PCR扩增的机会。 我们建议在福尔马林固定后清洗粪便标本，并将清洗后的标本储存在磷酸盐缓冲盐水中。我们开发了一种更短、更便宜、更简单的从粪便中提取微孢子虫DNA的方法。