The development of FastMycoâ¢ : A novel isothermal colorimetric assay for the rapid detection of mycoplasma contamination .

FastMyco™ 的开发：一种用于快速检测支原体污染的新型等温比色测定法。

基本信息

批准号：
10080974
负责人：
DEAN ROSENTHAL
金额：
$ 27.43万
依托单位：
AMELIA TECHNOLOGIES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10080974
关键词：
Acholeplasma laidlawii Address Adopted Agriculture Alkalies Arginine Binding Biological Biological Assay Biomedical Research Cell Culture Techniques Cell Line Cell Maintenance Cell Wall Cell membrane Color Coupled Culture Media Cytolysis DNA Decision Making Detection Detergents Development Diagnostic tests Dyes Enzymes Equipment Exhibits Fluorescence Freeze Drying Gentian Violet Hand Human Incubators Individual Infrastructure Laboratories Link Magnesium Major Groove Mammalian Cell Medical Research Methods Modernization Mycoplasma Mycoplasma hyorhinis Organism Output Perforation Phase Polymerase Prevalence Price Protocols documentation RNA RNA amplification RNA-Directed DNA Polymerase Reaction Reagent Reporting Reproducibility Research Ribosomal RNA Sampling Scientist Ships Small Business Innovation Research Grant Sodium Chloride Sodium Hydroxide Source Speed Spottings System Techniques Temperature Testing Time Tube United States National Institutes of Health Viola amplification detection aqueous assay development base biological research commercial application computerized data processing cost design experimental study innovation luminescence novel novel strategies pathogen pathogenic bacteria pathogenic virus prototype rapid detection recombinase remote location research facility tissue culture triphenylmethane

项目摘要

SBIR 2020: 6823823214: The development of FastMyco™: A novel isothermal colorimetric assay for the rapid detection of mycoplasma contamination. The development of FastMyco™: A novel isothermal colorimetric assay for the rapid detection of mycoplasma contamination. Project Summary/ Abstract (Word Count: 350) Mycoplasma (myco) contamination of mammalian cell lines are recognized as a major contributor to the lack of reproducibility in modern biomedical research. Despite the availability of commercially marketed detection systems, the unabated prevalence of myco in research attests to the shortcomings of these protocols. Indeed, all currently marketed myco tests fail to address the actual needs of the consumer, the bench scientist. For a myco detection product to be widely adopted it must: 1) require no additional equipment or infrastructure, 2) integrate seamlessly into a laboratory’s daily tissue culture routine with no additional labor burden on the users, and 3) be sensitive, inexpensive, and yield results immediately. To this end, in Phase I of this proposal we will optimize the FastMyco™ assay to test cell-culture media samples. FastMyco™ uses breakthrough Recombinase Polymerase Amplification (RPA) coupled to an initial reverse transcriptase (RT) step (RT-RPA), resulting in an all-in-one isothermal alternative to PCR myco detection. In FastMyco™, the RT-RPA reaction will target the 16S rRNA of myco, with amplification conducted in the cell-culture incubator at 37°C for approx. 15- 20 minutes. The assay has a potential detection limit of less than 1 CFU/ ml and immediate colorimetric output from an onboard detection system. The thermal stability of the system is a major technical and cost advantage of FastMyco™ over comparable systems. This will be achieved using cutting-edge lyophilization techniques that will create a multi-layered bead containing all necessary enzymes and reagents, freeze-dried around a core of DNA-detection substrate. The bead dissolves at different rates releasing the detection reagent only after target amplification has begun. In Phase II, we will continue to develop FastMyco™ for large HTP research organizations but also expand the bead-based detection systems to other human and agricultural pathogens. We will strive to integrate FastMyco™ into every laboratory’s routine cell-culture. The assay would also give regulatory agencies such as NIH and FDA the leverage to require this more rigorous testing protocols to help curb the spread of mycoplasma in research.

SBIR 2020：6823823214：FastMyco™ 的开发：一种用于快速检测支原体污染的新型等温比色测定法。 FastMyco™ 的开发：一种用于快速检测的新型等温比色测定法支原体污染。项目概要/摘要（字数：350）哺乳动物细胞系的支原体 (myco) 污染被认为是导致缺乏尽管有商业化的检测，但现代生物医学研究的可重复性。事实上，研究中真菌的流行有增无减，证明了这些方案的缺点。目前销售的所有真菌测试都无法满足消费者（即实验室科学家）的实际需求。为了使真菌检测产品得到广泛采用，它必须：1) 不需要额外的设备或基础设施， 2) 无缝集成到实验室的日常组织培养程序中，不会给实验室带来额外的劳动负担用户，以及 3) 敏感、成本低廉，并立即产生结果为此，在本提案的第一阶段。我们将优化 FastMyco™ 测定法来测试细胞培养基样品 FastMyco™ 使用突破性的技术。重组酶聚合酶扩增 (RPA) 与初始逆转录酶 (RT) 步骤 (RT-RPA) 偶联，从而成为 FastMyco™ 中 RT-RPA 反应的一种一体化等温替代方案。将靶向 myco 的 16S rRNA，在细胞培养箱中于 37°C 下进行约 3 次扩增。该测定的潜在检测限低于 1 CFU/ml，并可立即进行比色。系统的热稳定性是一个主要的技术和成本。 FastMyco™ 相对于同类系统的优势这将通过尖端的冻干技术来实现。技术将产生包含所有必要的酶和试剂的多层珠子，冷冻干燥围绕 DNA 检测基质的核心，珠子以不同的速率溶解，释放检测结果。仅在目标扩增开始后才使用试剂在第二阶段，我们将继续开发用于大型的 FastMyco™。 HTP 研究组织还将基于微珠的检测系统扩展到其他人类和我们将努力将 FastMyco™ 融入每个实验室的常规细胞培养中。检测还将使 NIH 和 FDA 等监管机构有能力要求更严格的检测测试方案有助于遏制支原体在研究中的传播。