Phage-Enabled Lab-on-a-Filter for Pathogen Separation, Concentration, and Detection

用于病原体分离、浓缩和检测的噬菌体实验室过滤器

基本信息

批准号：
9920143
负责人：
Sam R Nugen
金额：
$ 18.82万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-15 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9920143
关键词：
Address Adopted Adsorption Affinity Antigens Bacteria Bacterial Infections Bacteriophage T4 Bacteriophages Binding Biological Assay Biosensor Carbohydrates Cellular Phone Cellulose Clinical Colony-forming units Color Combating Antibiotic Resistant Bacteria Custom Cytolysis DNA Detection Development Diagnostic Discrimination Distal Dyes Engineering Enzyme Reactivation Enzymes Equipment Escherichia coli Escherichia coli O157:H7 Fiber Genes Genetic Engineering Goals Health Host resistance Hour Human Immobilization Immobilized Enzymes Infection Knowledge Lead Life Cycle Stages Liquid substance Location Magnetism Mediating Methods Multi-Drug Resistance Mutation Nanotechnology Optics Outcome Pathogen detection Performance Precipitation Preparation Process Protocols documentation Reaction Reporter Reporter Genes Reporting Research Resistance Safety Salmonella Sampling Sodium Chloride Stains Structural Protein Surface Surface Antigens Tail Techniques Technology Temperature Time Virus Visual Water Work base combat cost design diagnostic assay medical food meetings multiplex detection new technology overexpression pathogen pathogenic bacteria rapid detection receptor sensor synthetic biology tool

项目摘要

Project Summary While new technologies for detecting pathogens are often reported, these typically require small volumes of concentrated and clean samples which can make them impractical to use. The long-term goal is to develop pragmatic, low-cost and easy-to-use assays to identify, separate, concentrate, and detect low concentrations of target bacteria in liquid samples The objective of this application is to use synthetic biology to overcome current obstacles in phage-based detection including sensor performance and host resistance. Two specific aims have been developed towards this objective, 1) Engineer an E. coli-specific phage to produce a cellulose- binding reporter enzyme to enable a “Lab on a Filter” detection assay, and 2) Engineering bacteriophages to avoid host resistance. By considering a filter to be a reaction surface, a “Lab on a Filter” concept which can rapidly reduce the time to results and provide low concentration quantification of bacteria in enabled. Bacteriophages (phages) are viruses which infect bacteria, and can be engineered to deliver genes for reporter enzymes to target filtered bacteria during an assay. The enzymes would be overexpressed and released by the bacterial host during the infection. Enzymes fused with a cellulose-binding module would immobilize directly on a cellulose filter in proximity to the lysed bacteria. Enzyme-reactive precipitating dyes can then be used to form colored precipitate in the proximity of the immobilized enzymes. The result is a fully quantitative (0 – 250 CFU/100 mL) and assay for bacteria which is amenable to both standard and non-laboratory settings and can be provide results after only a few hours. Phages which target and kill specific bacteria exist for almost all known bacterial pathogens. The use of phages for both bacteria detection and for combating multidrug resistant bacterial infections continues to increase. The main hurdle with using phages for this purpose, is the ability of the bacterial host to evolve resistance through random mutations of surface antigens. The ability to genetically engineer phages to avoid host resistance will have a significant and positive impact on phage- based pathogen detection as well as phage therapy to treat multidrug-resistant-bacterial infections. By engineering a phage to have multiple surface recognition receptors (tail fibers), the bacterial host would require several mutations to avoid adsorption of the phages. This can be performed by engineering mixed tail fibers targeting the same pathogen into one phage. In addition, a phage will be engineered that contains mixed tail fibers specific to Salmonella and E. coli to demonstrate the engineering of the phages' host range. The proposed research is significant because while phages have evolved to be near perfect predators of specific bacteria, practical hurdles have limited their use for pathogen detection and treatment. By mitigating these hurdles, significant advances toward human health and safety can be achieved using genetically engineered phages.

项目摘要尽管经常报告用于检测病原体的新技术，但通常需要少量浓缩和干净的样品可能使它们不切实际。长期目标是发展务实，低成本和易于使用的测定法，以识别，分离，浓缩和检测低浓度液体样品中靶细菌的目的是使用合成生物学克服基于噬菌体的检测中的当前障碍物，包括传感器性能和宿主电阻。两个具体目标是针对这个目标的，1）工程师一种大肠杆菌特异性的噬菌体来产生纤维素 - 结合记者酶以实现“在过滤器上的实验”检测测定法，以及2）工程细菌至避免宿主阻力。通过将过滤器视为反应表面，“滤波器上的实验室”概念可以快速减少结果的时间，并提供启用细菌的低浓度定量。噬菌体（噬菌体）是感染细菌的病毒，可以设计为为记者提供基因在测定过程中靶向过滤细菌的酶。这些酶将过表达并释放感染期间细菌宿主。与纤维素结合模块融合的酶会固定直接在纤维素过滤器上，靠近粗糙的细菌。然后可以是酶反应性沉淀染料用于在固定酶的接近度中形成有色沉淀物。结果是完全定量的（0 - 250 CFU/100 mL）和对细菌的测定，这是标准和非实验室设置仅几个小时后就可以提供结果。几乎存在靶向和杀死特定细菌的噬菌体所有已知的细菌病原体。使用噬菌体来检测细菌和对抗多药抗性细菌感染继续增加。为此目的使用噬菌体的主要障碍是细菌宿主通过表面抗原的随机突变发展抗性的能力。能力遗传工程师噬菌体以避免宿主抵抗力将对噬菌体产生重大和积极的影响 - 基于病原体检测以及噬菌体治疗，以治疗多药抗性细菌感染。经过工程噬菌体具有多个表面识别受体（尾纤维），细菌宿主需要几个突变以避免噬菌体增加。这可以通过工程混合尾纤维来执行将相同的病原体靶向一个噬菌体。此外，将设计一个包含混合尾巴的噬菌体特定于沙门氏菌和大肠杆菌的纤维以证明噬菌体宿主范围的工程。这拟议的研究很重要，因为尽管噬菌体已经发展为几乎是特定的完美捕食者细菌，实际障碍限制了它们用于病原体检测和治疗的用途。通过减轻这些障碍，可以使用基因工程来实现人类健康和安全的重大进步噬菌体。