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：一种新型的等温比色测定法，用于快速检测支原体污染。 FastMyco™的开发：一种新颖的等温比色测定，用于快速检测支原体污染。项目摘要/摘要（单词计数：350）哺乳动物细胞系的支原体（Myco）污染被认为是缺乏的主要因素现代生物医学研究中的可重复性。尽管有商业销售的检测可用系统，在研究中，迈克的流行率没有减弱，这证明了这些协议的缺点。的确，当前所有销售的Myco测试都无法满足消费者，替补席科学家的实际需求。为了广泛采用Myco检测产品：1）不需要其他设备或基础设施， 2）无缝集成到实验室的日常组织培养程序中，没有其他实验室伯恩。用户和3）立即保持敏感，廉价和产量结果。为此，在本提案的第一阶段我们将优化FastMyCo™测定法以测试细胞培养培养基样品。 FastMyco™使用突破重点酶聚合酶扩增（RPA）耦合到初始逆转录酶（RT）步骤（RT-RPA），导致PCR Myco检测的多合时等温替代方法。在FastMyco™中，RT-RPA反应将靶向迈克的16S rRNA，并在37°C的细胞培养孵化器中进行扩增。 15-20分钟。该测定的潜在检测极限小于1 cfu/ ml，立即进行比色学从车载检测系统输出。系统的热稳定性是主要的技术和成本 FastMyco™优于可比较系统的优势。这将使用尖端的冻干来实现将创建一个多层珠的技术，其中包含所有必要的酶和试剂，冷冻干燥 DNA检测底物的核心周围。珠子以不同的速率溶解以释放检测只有在目标扩增之后才开始进行试剂。在第二阶段，我们将继续开发大型的FastMyco™ HTP研究组织，但也将基于珠子的检测系统扩展到其他人类和农业病原体。我们将努力将FastMyco™集成到每个实验室的常规细胞文化中。这测定还将为NIH和FDA等监管机构提供更严格的杠杆作用测试方案有助于遏制支原体在研究中的传播。