DIAGNOSTICS FOR MICROSPORIDIA

微孢子虫的诊断

基本信息

批准号：
7958699
负责人：
Elizabeth Schmidt Didier
金额：
$ 5.8万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7958699
关键词：
Betaine Bile fluid Buffers Cells Clinical Computer Retrieval of Information on Scientific Projects Database Computer software Detection Diagnosis Diarrhea Enterocytozoon bieneusi Enzymes Equipment Funding Genbank Gene Targeting Grant Hour Human Immune Infection Institution Invertebrates Laboratories Macaca mulatta Manuals Methods Microsporidia Microsporidiosis Molecular Nested PCR Parasites Polymerase Primates Reproduction spores Research Research Personnel Resources Sensitivity and Specificity Septata intestinalis Source Special Equipment Specificity Specimen Systemic disease Time United States National Institutes of Health Work age group base cost field study improved rRNA Genes tissue culture

Betaine Bile fluid Buffers Cells Clinical Computer Retrieval of Information on Scientific Projects Database Computer software Detection Diagnosis Diarrhea Enterocytozoon bieneusi Enzymes Equipment Funding Genbank Gene Targeting Grant Hour Human Immune Infection Institution Invertebrates Laboratories Macaca mulatta Manuals Methods Microsporidia Microsporidiosis Molecular Nested PCR Parasites Polymerase Primates Reproduction spores Research Research Personnel Resources Sensitivity and Specificity Septata intestinalis Source Special Equipment Specificity Specimen Systemic disease Time United States National Institutes of Health Work age group base cost field study improved rRNA Genes tissue culture

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项目摘要

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). Control Ent. bieneusi and Enc. intestinalis spores were obtained from rhesus macaque bile and tissue culture supernatants, respectively, and 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. Lower concentrations of betaine or higher concentrations of dNTPs typically generated negative results. The advantages of the accelerated LAMP over standard and nested PCR include lower cost, shorter time, higher specificity, and no requirement for special equipment. Accelerated LAMP, however, was approximately 10-fold less sensitive than nested PCR. These results support continued work to improve the sensitivity of accelerated LAMP to more efficiently detect microsporidia-positive specimens in clinical laboratory and field studies.

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）。控制ENT。 Bieneusi和Enc。 intestinalis spores were obtained from rhesus macaque bile and tissue culture supernatants, respectively, and 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酶在80¿C中停用。C。较低的甜菜碱浓度或更高浓度的DNTP通常会产生负面结果。加速灯的优点比标准和嵌套的PCR的优点包括较低的成本，较短的时间，更高的特异性以及对特殊设备的不需要。然而，加速灯比嵌套PCR敏感约10倍。这些结果支持继续工作，以提高加速灯的敏感性，以更有效地检测临床实验室和现场研究中的微孢子虫阳性标本。