Multiplexed Detection of Bioterror Agents

生物恐怖分子的多重检测

• 批准号: 6845582
• 负责人: FRANCIS BARANY
• 金额: $ 444.88万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845582
• 关键词: antibiotics; biohazard control; biohazard detection; bioterrorism /chemical warfare; capillary electrophoresis; clinical research; cooperative study; drug resistance; gel electrophoresis; high throughput technology; human tissue; microbiology; polymerase chain reaction; septicemia; virulence

DESCRIPTION (provided by applicant): The current biothreat to our nation requires the ability to rapidly detect and distinguish bioweapon agents from normal pathogens. Existing detection systems have a limited ability to simultaneously screen in a single sample for multiple agents and their antibiotic resistance, toxin, or virulence genes. To meet this need we propose to use ligase detection reaction (LDR) techniques combined with PCR, capillary electrophoresis, and Universal Arrays, which we have already validated in the detection of cancer gene mutations and the diagnosis of genetic diseases. The studies will include the evaluation of Quantum-dots (Q-dots) as a novel detection approach. The above techniques will be used to meet the specific aims of this proposal: Aim 1: To identify multiple blood-borne pathogens simultaneously, in a single assay. Samples will be evaluated for 16 common bacterial pathogens as well as four blood-borne bioterror agents (B. anthracis, Y. pestis, F. tularensis, B. abortus). Blood specimens from patients with suspected bacteremia will be evaluated in the microbiology laboratory and tested using the above molecular techniques. Random samples will also be spiked with DNA of bioterror pathogens to simulate infection with these agents. Likewise, HIV RNA will be used as a surrogate for RNA hemorrhagic fever viruses. Aim 2: To evaluate antibiotic resistance, enhanced virulence, or genetic manipulation in a positive blood culture. LDR/PCR will be used to detect the presence of the vanA, vanB, mecA, tetL, tetM, gyrA and grlA mutations, additional antibiotic resistance determinants, virulence and toxin genes, which may be present in the common blood-borne pathogens identified in Aim 1. Aim 3: To distinguish Category ABC, engineered, or emergent biothreat agents from common pathogens employing high throughput screening platforms using microbial signature profiles. Standardized protocols using common liquid handling robotic platforms will be established using the techniques validated in Aims 1 and 2. The test will be validated for detecting the above pathogens and further viral agents: Ebola, Marburg, Lassa, Crimean-Congo, Hemorrhagic Fever, Rift Valley Fever, Yellow Fever, SARS Coronavirus, Dengue, and West Nile virus, as well as to distinguish Variola from Vaccinia. In addition, the LDR/PCR virulence gene test from Aim 2 will be expanded to include the major BT toxin and virulence genes. Once verified, our tests will be validated with clinical samples at the CDC.

描述（由申请人提供）：目前对我国的生物威胁需要快速检测和区分生物武器制剂与正常病原体的能力。现有的检测系统在单一样品中同时筛选多种试剂及其抗生素抗性、毒素或毒力基因的能力有限。为了满足这一需求，我们建议使用连接酶检测反应（LDR）技术结合PCR，毛细管电泳和通用阵列，我们已经验证了在检测癌症基因突变和遗传性疾病的诊断。这些研究将包括评估量子点（Q-dots）作为一种新的检测方法。上述技术将用于满足本提案的具体目标：目标1：在一次检测中同时鉴定多种血源性病原体。将对样本进行16种常见细菌病原体以及4种血源性生物恐怖剂（B）的评价。anthracis，Y.鼠疫F.土拉菌，B. tularensis，B.流产）。疑似菌血症患者的血液标本将在微生物学实验室进行评价，并使用上述分子技术进行检测。随机样本中还将掺入生物恐怖病原体的DNA，以模拟这些病原体的感染。同样，HIV RNA将被用作RNA出血热病毒的替代物。目的2：评估阳性血培养中的抗生素耐药性、增强的毒力或遗传操作。LDR/PCR将用于检测目标1中确定的常见血液传播病原体中是否存在vanA、vanB、mecA、tetL、tetM、gyrA和grlA突变、其他抗生素耐药决定簇、毒力和毒素基因。目标三：采用高通量筛选平台，使用微生物特征谱区分ABC类、工程或紧急生物威胁因子与常见病原体。将使用目标1和2中确认的技术建立使用通用液体处理机器人平台的标准化方案。将验证该试验用于检测上述病原体和其他病毒因子：埃博拉、马尔堡、拉沙、克里米亚-刚果、出血热、裂谷热、黄热病、SARS冠状病毒、登革热和西尼罗河病毒，以及区分天花和牛痘。此外，目标2中的LDR/PCR毒力基因检测将扩展至包括主要的BT毒素和毒力基因。一旦得到验证，我们的测试将在CDC进行临床样本验证。

项目成果

Ligase detection reaction generation of reverse molecular beacons for near real-time analysis of bacterial pathogens using single-pair fluorescence resonance energy transfer and a cyclic olefin copolymer microfluidic chip.

• DOI: 10.1021/ac101843n
• 发表时间: 2010-12-01
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Peng, Zhiyong;Soper, Steven A.;Pingle, Maneesh R.;Barany, Francis;Davis, Lloyd M.
• 通讯作者: Davis, Lloyd M.

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

用于同时鉴定A类感染性病原体的多重PCR/LDR分析：病毒出血热和Variola病毒的药物。

• DOI: 10.1371/journal.pone.0138484
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Das S;Rundell MS;Mirza AH;Pingle MR;Shigyo K;Garrison AR;Paragas J;Smith SK;Olson VA;Larone DH;Spitzer ED;Barany F;Golightly LM
• 通讯作者: Golightly LM

A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

• DOI: 10.1038/s41598-018-30301-3
• 发表时间: 2018-09-04
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Mirza AH;Das S;Pingle MR;Rundell MS;Armah G;Gyan B;Hodinka RL;Larone DH;Spitzer ED;Barany F;Golightly LM
• 通讯作者: Golightly LM