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）：该项目的目标是利用手持式、易于使用、廉价、封闭的完全集成核酸检测设备，促进初级卫生保健机构在护理点从痰液中快速诊断耐药结核病设备。与目前用于护理点核酸检测的基于 PCR 的完全集成台式系统相比，设想的系统的成本将降低 10-100 倍，尺寸也将显着缩小。该项目建立在正在进行的工作基础上，旨在开发一种类似但更简单的系统，用于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) 与侧流视觉检测相结合。简单快速的样品制备利用最初为机械裂解炭疽孢子而开发的小型珠子混合器，其当前版本还能够进行核酸纯化。所提出的概念涉及一种手持式电池供电仪器，其上附有一次性塑料盒。该盒包含用于样品制备、扩增和检测的室、阀门和流体导管，所有这些都在一个封闭的系统中。在其最终形式中，试剂盒将包含机上所有必需的试剂。手持式仪器控制测试执行所需的泵送、阀门和等温加热步骤。操作员只需将试剂盒连接到仪器上，插入样品，按下按钮即可启动测试，大约 30 分钟到一小时后读取结果。这样的系统将允许医疗保健专业人员在现场护理环境中及时诊断和治疗耐药结核病，从而改善患者的治疗结果并防止进一步传播。该项目的目标1重点是建立一种简单、快速、有效的适用于痰液中结核分枝杆菌的样品制备方法。在目标 2 下，我们将建立基于 EXPAR 的多重等温核酸扩增测定，并结合基于侧流的检测。该项目的目标 3 侧重于开发卡盒和仪器原型，以执行目标 1 和 2 下开发的样品制备、等温扩增和视觉检测方法。目标 1-3 中描述的任务的性能验证最初将涉及 BSL2 替代物和 DNA 提取物结核分枝杆菌，然后是在培养物中生长并掺入结核分枝杆菌阴性痰中的药物敏感和耐药结核分枝杆菌，最后是经过分枝杆菌检测的临床痰样本。使用既定方法测定结核病和抗菌药物敏感性。该项目提供了一个独特的机会，可以汇集多学科专业知识来开发下一代核酸检测方法和分析仪。从长远来看，本文开发的测定和设备可应用于与资源有限环境中的生物威胁检测和传染病诊断相关的一系列病原体。该项目的目标是利用手持式、易于使用、廉价、封闭单元的完全集成核酸检测设备，促进初级卫生保健机构在护理点通过痰液快速诊断耐药结核病。与目前用于护理点核酸检测的基于 PCR 的完全集成台式系统相比，设想的系统的成本将降低 10-100 倍，尺寸也将显着缩小。

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.