Rapid Diagnosis of MDR TB from Sputum using a Small Fully Integrated Nucleic Acid

使用小型完全集成核酸从痰中快速诊断耐多药结核病

基本信息

批准号：
8493985
负责人：
Angelika Niemz
金额：
$ 80.31万
依托单位：
KECK GRADUATE INST OF APPLIED LIFE SCIS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8493985
关键词：
Address Antimicrobial susceptibility Bacillus anthracis spore Biological Assay Biomedical Research Clinic Clinical Collaborations Complex County Coupled Cytolysis DNA Detection Development Devices Diagnosis Diagnostic Drug Resistant Tuberculosis Drug resistance Drug resistance in tuberculosis Drug usage Engineering Genus Mycobacterium Goals Grant Health Professional Health care facility Heating Hour Housing Human Incidence Institutes Intervention Laboratories Lateral Liquid substance Mechanics Methods Microfluidics Multidrug-Resistant Tuberculosis Mycobacterium tuberculosis Nucleic Acid Amplification Tests Nucleic Acids Outcome Patients Performance Pharmaceutical Preparations Phase Philippines Plastics Point-of-Care Systems Population Predisposition Preparation Primary Health Care Publications Pump Reaction Reading Reagent Research Institute Resistance Resources Rifampicin resistance Sampling Science Simplexvirus Solid Specimen Sputum Staging Swab System Technology Testing Thick Time Validation Variant Visual Washington Work base biothreat communicable disease diagnosis cost design design and construction effective therapy flexibility global health improved instrument internal control manufacturing scale-up meetings miniaturize multidisciplinary next generation nucleic acid purification pathogen point of care pre-clinical prevent prototype rapid diagnosis resistance mutation viral DNA

项目摘要

DESCRIPTION (provided by applicant): The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing. The project builds upon ongoing work aimed at developing a similar albeit simpler system for detection of Herpes Simplex Virus from swab samples. The assay is based on the isothermal EXponential Amplification Reaction (EXPAR) coupled to visual detection using lateral flow. Simple and rapid sample preparation utilizes a miniaturized bead blender originally development for mechanical lysis of anthrax spores, which in its current version is also capable of nucleic acid purification. The proposed concept involves a handheld, battery operated instrument, to which a disposable plastic cartridge is attached. The cartridge houses chambers, valves, and fluid conduits for sample preparation, amplification and detection, all in a single, closed system. In its final form, the cartridge will contain all necessary reagents on board. The handheld instrument controls the pumping, valving and isothermal heating steps necessary for test execution. The operator simply attaches the cartridge to the instrument, inserts the sample, initiates the test by pushing a button, and after approximately 30 minutes to one hour reads off the result. Such a system will allow health care professionals in a point-of- care setting to diagnose and treat drug resistant tuberculosis in a timely manner, thereby improving patient outcomes and preventing further spread. Aim 1 of this project focuses on establishing a simple, rapid and effective sample preparation method applicable to Mycobacterium tuberculosis in sputum. Under Aim 2, we will establish multiplexed isothermal nucleic acid amplification assays based on EXPAR, coupled with lateral flow based detection. Aim 3 of this project focuses on developing cartridge and instrument prototypes to execute the sample preparation, isothermal amplification, and visual detection methods developed under aims 1 and 2. Performance verification for tasks described under Aims 1-3 will initially involve BSL2 surrogates and DNA extracts of M. tuberculosis, followed by drug susceptible and resistant M. tuberculosis grown in culture and spiked into TB negative sputum, and ultimately clinical sputum samples that have been tested for M. tuberculosis and antimicrobial susceptibility using established methods. This project presents a unique opportunity to bring together multidisciplinary expertise to develop a next generation of assays and analyzers for nucleic acid testing. In the long term, the assays and devices developed herein can be applied to a spectrum of pathogens relevant to biothreat detection and infectious disease diagnosis in resource-limited settings. The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing.

描述（由申请人提供）：该项目的目的是使用手持式，易于使用的，易于使用，廉价，封闭式完全集成的核酸测试装置的核心设施中的护理点快速诊断出痰液中的抗药性结核病。设想系统的成本将低10-100倍，并且大小将明显小于基于PCR的完全集成的台式系统，用于护理核酸测试。该项目建立在正在进行的工作的基础上，旨在开发类似的简单系统，尽管可以从拭子样本中检测单纯疱疹病毒。该测定基于等温指数扩增反应（Expar）与横向流相结合的视觉检测。简单而快速的样品制备利用了最初开发的微型珠搅拌器来开发炭疽孢子的机械裂解，在其当前版本中，它也能够进行核酸纯化。提出的概念涉及一个手持电池操作的仪器，将一次性塑料盒附着在该仪器上。墨盒容纳腔室，阀门和流体导管，用于样品制备，放大和检测，全部在一个封闭的系统中。在最终形式中，该墨盒将包含船上所有必要的试剂。手持仪器控制测试执行所需的泵送，阀门和等温加热步骤。操作员只需将墨盒连接到仪器上，插入样品，通过按下按钮来启动测试，然后大约30分钟到一小时后读取结果。这样的系统将允许在护理点环境中的医疗保健专业人员及时诊断和治疗耐药性结核病，从而改善患者的结局并防止进一步扩散。该项目的目标1着重于建立一种适用于痰液中结核分枝杆菌的简单，快速和有效的样品制备方法。在AIM 2下，我们将基于Expar建立多路复用的等温核酸扩增测定法，并结合基于侧流的检测。该项目的目标3着重于开发弹药和仪器原型，以执行样品制备，等温度扩增以及目标1和2下开发的视觉检测方法。目的1-3所描述的任务的性能验证最初将涉及BSl2最终使用已建立的方法测试了针对结核分枝杆菌和抗菌敏感性测试的临床痰样品。该项目提供了一个独特的机会，将多学科专业知识汇总在一起，以开发下一代的测定法和分析仪进行核酸测试。从长远来看，本文开发的分析和设备可以应用于与资源有限的环境中与生物治疗检测和传染病诊断有关的一系列病原体。该项目的目的是使用手持式，易于使用，易用，廉价，封闭的单元完全集成的核酸测试装置来促进初级保健设施中痰液中抗药性结核病的快速诊断。设想系统的成本将低10-100倍，并且大小将明显小于基于PCR的完全集成的台式系统，用于护理核酸测试。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.

DOI：
10.1038/srep19541
发表时间：
2016-01-20
期刊：
Scientific reports
影响因子：
4.6
作者：
Ferguson TM;Weigel KM;Lakey Becker A;Ontengco D;Narita M;Tolstorukov I;Doebler R;Cangelosi GA;Niemz A
通讯作者：
Niemz A

Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.

DOI：
10.1039/d0lc00445f
发表时间：
2020-10-27
期刊：
Lab on a chip
影响因子：
6.1
作者：
Lu HW;Sakamuri R;Kumar P;Ferguson TM;Doebler RW;Herrington KD;Talbot RP;Weigel KM;Nguyen FK;Cangelosi GA;Narita M;Boyle DS;Niemz A
通讯作者：
Niemz A

Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries.