DIAGNOSTICS FOR MICROSPORIDIA

微孢子虫的诊断

• 批准号: 8173013
• 负责人: Elizabeth Schmidt Didier
• 金额: $ 6.18万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173013
• 关键词: Betaine; Bile fluid; Buffers; Cells; Computer Retrieval of Information on Scientific Projects Database; Computer software; Culture Media; DNA; Detection; Development; Diagnosis; Diagnostic; Diarrhea; Enterocytozoon bieneusi; Enzymes; Equipment; Feces; Funding; Genbank; Gene Targeting; Grant; Hour; Human; Immune; Infection; Institution; Invertebrates; Macaca mulatta; Manuals; Methods; Microsporidia; Microsporidiosis; Molecular; Nested PCR; Paper; Parasites; Polymerase; Reaction; Recombinant DNA; Reproduction spores; Research; Research Personnel; Resources; Sampling; Sensitivity and Specificity; Septata intestinalis; Source; Special Equipment; Specificity; Specimen; Systemic disease; Testing; Time; United States National Institutes of Health; age group; base; cost; improved; inhibitor/antagonist; rRNA Genes; sample fixation; tissue culture

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microsporidia are obligately intracellular, single-celled fungal parasites that infect invertebrate and vertebrate hosts. Infections due to microsporidia occur worldwide in both immune-deficient and immune-competent humans of all age groups and are associated with diarrhea and systemic disease. Molecular-based PCR methods have improved sensitivity and specificity for diagnosing microsporidiosis but require costly equipment (eg. thermocycler) and take hours to perform. In this study, an accelerated LAMP method was applied to improve the time efficiency for molecular detection of the two most prevalent microsporidia species infecting humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis. Primer Explorer LAMP software (http://primerexplorer.jp/e/v3_manual/index.html) was used to select primers from the ribosomal RNA gene targets of Ent. bieneusi (Genbank L07123) and Enc. intestinalis (Genbank L19567). Specimens tested included Ent. bieneusi and Enc. intestinalis spores obtained from rhesus macaque bile and tissue culture supernatants that were suspended in tissue culture medium or spiked into stool. Extracted DNA samples were reacted with 0.8 uM FIP and BIP primers, 0.2 uM F3 and B3 primers, 0.4 uM LF and LB loop primers, 1.5 M betaine, 16 units Bst polymerase, and 0.5 M dNTPs in Bst buffer at a final volume of 50 ul for 1 hour at 63¿ C followed by 10 min enzyme deactivation at 80¿ C for the LAMP or via nested PCR using rDNA primers. Sensitivity of nested PCR was 1  10 spores per reaction volume (or 10  100 spores / ml feces). LAMP was a log less sensitive at 10  100 spores per reaction volume or 100  1000 spores per ml feces. Attempts are in progress to improve sensitivity of LAMP using FTA Whatman filter papers for fixation of microsporidia DNA prior to extraction and amplification in attempt to remove inhibitors. These results support further development of LAMP due to its lower cost, shorter time, higher specificity, and no requirement for special equipment when compared to nested PCR, but sensitivity of detection with LAMP needs to be improved.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 微孢子虫是一种专性细胞内单细胞真菌寄生虫，可感染无脊椎动物和脊椎动物宿主。由微孢子虫引起的感染在世界范围内的所有年龄段的免疫缺陷和免疫功能正常的人类中都有发生，并且与腹泻和基于分子的 PCR 相关。这些方法提高了诊断微孢子虫病的灵敏度和特异性，但需要昂贵的设备（例如热循环仪）并且需要数小时才能完成。应用加速 LAMP 方法来提高感染人类的​​两种最流行的微孢子虫——比氏肠细胞虫和肠脑孢子虫 Primer Explorer LAMP 软件的时间效率 (http://primerexplorer.jp/e/v3_manual/index.html)。用于从 Ent bieneusi (Genbank L07123) 和 Enc. 的核糖体 RNA 基因靶标中选择引物。 (Genbank L19567)。测试的样本包括从恒河猴胆汁和组织培养上清液中获得的 Ent. bieneusi 和 Enc. 孢子，这些孢子悬浮在组织培养基中或掺入粪便中，与 0.8 uM FIP 和 BIP 引物反应。 0.2 uM F3 和 B3 引物，0.4 uM LF 和 LB 环引物， 1.5 M 甜菜碱、16 单位 Bst 聚合酶和 0.5 M dNTP 在 Bst 缓冲液中，最终体积为 50 ul，在 63° 下反应 1 小时C，然后在 80°C 下酶失活 10 分钟C 对于 LAMP 或通过使用 rDNA 引物的巢式 PCR，巢式 PCR 的灵敏度为每反应体积 1 10 个孢子（或每毫升粪便 10 100 个孢子），LAMP 在每反应体积 10 100 个孢子或 100 1000 个孢子时灵敏度较低。正在尝试使用 FTA 提高 LAMP 的灵敏度。 Whatman 滤纸用于在提取和扩增之前固定微孢子虫 DNA，以试图去除抑制剂。这些结果支持 LAMP 的进一步开发，因为与巢式 PCR 相比，其成本更低、时间更短、特异性更高，并且不需要特殊设备。但LAMP检测的灵敏度有待提高。