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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 微孢子虫基本上是细胞内的单细胞真菌寄生虫，感染了无脊椎动物和脊椎动物宿主。由于微孢子虫引起的感染在全世界都发生在所有年龄段的免疫缺陷和免疫能力的人类中，并且与腹泻和全身性疾病有关。基于分子的PCR方法具有提高诊断微孢子虫病的灵敏度和特异性，但需要昂贵的设备（例如热环生物）并需要小时才能进行。在这项研究中，采用了一种加速灯法来提高两个最普遍的微孢子虫物种感染的人类，肠肠球菌生物素和脑肠道肠道的时间效率。 Primer Explorer Lamp Software（http://primerexplorer.jp/e/v3_manual/index.html）用于从ENT的核糖体RNA基因靶标中选择引物。 Bieneusi（GenBank L07123）和ENC。肠道（GenBank L19567）。测试的标本包括ENT。 Bieneusi和Enc。从恒河猕猴胆汁和组织培养上清液中获得的肠道孢子，这些上清液悬浮在组织培养基中或尖刺成粪便。 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引物。嵌套PCR的敏感性为每个反应体积（或10 100孢子 / mL粪便）1 10个孢子。灯是对数较少敏感的，每个反应体积的10100孢子或每毫升粪便1001000个孢子。尝试使用FTA Whatman滤波纸来提高LAMP的灵敏度，以在提取和放大之前固定微孢子虫DNA，以尝试去除抑制剂。与嵌套PCR相比，这些结果支持灯的成本较低，时间较短，特异性较高，不需要特殊设备的要求进一步开发，但是需要提高对灯的检测敏感性。