A fully integrated assay and platform for detecting Clostridium difficile

用于检测艰难梭菌的完全集成的检测方法和平台

• 批准号: 7611310
• 负责人: Bruce D Irvine
• 金额: $ 31.12万
• 依托单位: CLAREMONT BIOSOLUTIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7611310
• 关键词: Alleles; Antibiotic Therapy; Bacteria; Biological Assay; Buffers; Calibration; Categories; Centrifugation; Clinic; Clinical; Clinical Research; Clostridium difficile; DNA; DNA Sequence; Decontamination; Detection; Development; Device or Instrument Development; Devices; Diagnosis; Diagnostic; Diarrhea; Dimensions; Disease; Disease remission; Drug Formulations; Environment; Environmental Pollution; Equipment; Feasibility Studies; Feces; Genes; Goals; Health; Health Care Costs; Hospital Nursing; Hour; Human; Infection; Israel; Judgment; Knowledge; Liquid substance; Maintenance; Manufacturer Name; Measures; Medical center; Methods; Morbidity - disease rate; Mutation; Nonsense Codon; Nursing Homes; Patients; Performance; Phase; Point Mutation; Point-of-Care Systems; Predisposition; Preparation; Process; Proteins; Reaction; Reagent; Rehabilitation therapy; Relative (related person); Reproduction spores; Risk; Sampling; Sensitivity and Specificity; Specimen; Swab; Syringes; System; Technology; Temperature; Testing; Time; Toxin; Training; Virulent; cost; cytotoxicity test; design; helicase; instrument; meetings; milliliter; mortality; mutant; point of care; premature; prevent; prospective; public health relevance

DESCRIPTION (provided by applicant): Antibiotic treatment of infections with harmful bacteria can destroy the normal colonic flora, and increase the patient's susceptibility to Clostridium difficile-associated disease (CDAD). Pathogenic strains of C. difficile commonly produce two large clostridial toxins, A (TcdA) and B (TcdB), responsible for CDAD. The TcdC gene is a negative regulator of the TcdA and TcdB genes, and the most highly virulent strain of C. difficile, BI/NAP1, has a single point mutation in this gene that results in a premature stop codon. The lack of a functional TcdC protein in these strains results in an over expression of the TcdA and TcdB toxins. This strain is associated with more severe morbidity and mortality in patients. Simple assays that are robust and specific for CDAD and in particular for the BI/NAP1 strain are needed to facilitate better diagnosis and treatment of C. difficile infected patients. The current, widely used, EIA tests are rapid but lack sensitivity whereas the more sensitive tests (cytotoxicity assay or C. difficile culture) require one or more days to complete. There is also clearly a need for a reliable, sensitive, and expeditious method for testing of environmental contamination. A sensitive assay that is capable of processing and detecting C. diff spores would substantially protect the health of the patient as well as reduce the risk of spread of CDAD in hospitals, nursing homes, and rehabilitation facilities. Most cases of remission are due to re-infection. Decontamination is very difficult and currently not supported by a reliable and sensitive method of verification. In this Phase I, we propose to develop a simple assay system for the detection of the TcdA gene, the TcdB gene and the TcdC point mutation using proprietary helicase-dependent amplification (HAD") technology. HDA reactions will detect TcdA for the presence and load of Clostridium difficile and TcdC D117 mutation for the presence of the highly virulent strain of C. difficile. We will develop a point-of-care (POC) instrument that will extract DNA from stool samples and environmental swabs. The sample processing methods selected for this plan are robust and simple, avoiding the need for wash steps, elution buffers, or centrifugation. This will be accomplished through the development of subsystem modules, each designed in a cartridge that is independently functional to its purpose, is disposable after one use, and can also be snapped together to the other modules for integrated function. This sample processing system will be integrated with the fluorimeter, a real-time fluorescent detection system that has been previously used with the HDA" assay method. PUBLIC HEALTH RELEVANCE: There is a clear clinical need for a rapid but sensitive test for C. difficile infection. The current, widely used, EIA tests are rapid but lack sensitivity whereas the more sensitive tests (cytotoxicity assay or C. difficile culture) require one or more days to complete. There is also clearly a need for a reliable, sensitive and expeditious method for testing for environmental contamination. A sensitive assay that is capable of processing and detecting C. diff spores would substantially protect the health of patients and reduce the cost of healthcare in hospitals, nursing homes and rehabilitation facilities. The goal of this project is to develop a point-of-care system for the detection of C. difficile.

描述（由申请人提供）：抗生素治疗有害细菌感染会破坏正常的结肠植物群，并增加患者对艰难梭菌相关疾病（CDAD）的易感性。病原菌株C.艰难梭菌通常产生两种大的梭菌毒素A（TcdA）和B（TcdB），其负责CDAD。TcdC基因是TcdA和TcdB基因的负调控基因，是C.艰难梭菌BI/NAP 1在该基因中具有导致提前终止密码子的单点突变。这些菌株中功能性TcdC蛋白的缺乏导致TcdA和TcdB毒素的过表达。该菌株与患者更严重的发病率和死亡率相关。为了更好地诊断和治疗C.艰难感染的患者。目前广泛使用的EIA测试是快速的，但缺乏灵敏度，而更敏感的测试（细胞毒性测定或C。艰难培养）需要一天或多天来完成。显然还需要一种可靠、灵敏和快速的方法来测试环境污染。一种灵敏的检测方法，能够处理和检测C。diff孢子将充分保护患者的健康，并降低CDAD在医院、疗养院和康复设施中传播的风险。大多数缓解病例是由于再次感染。清除污染非常困难，目前没有可靠和敏感的核查方法。在本阶段I中，我们提出使用专有的解旋酶依赖性扩增（HAD”）技术开发用于检测TcdA基因、TcdB基因和TcdC点突变的简单测定系统。HDA反应将检测TcdA以确定艰难梭菌的存在和载量，并检测TcdC D117突变以确定高毒力梭菌菌株的存在。很难我们将开发一种即时（POC）仪器，从粪便样本和环境拭子中提取DNA。本计划选择的样品处理方法耐用且简单，无需清洗步骤、洗脱缓冲液或离心。这将通过子系统模块的开发来实现，每个子系统模块都设计在一个盒中，该盒具有独立的功能，在一次使用后是一次性的，并且还可以与其他模块卡在一起以实现集成功能。该样品处理系统将与荧光计集成，荧光计是一种实时荧光检测系统，先前已与HDA”测定方法一起使用。 公共卫生相关性：临床上显然需要一种快速但敏感的C。艰难感染目前广泛使用的EIA测试是快速的，但缺乏灵敏度，而更敏感的测试（细胞毒性测定或C。艰难培养）需要一天或多天来完成。显然还需要一种可靠、灵敏和快速的方法来测试环境污染。一种灵敏的检测方法，能够处理和检测C。diff孢子将大大保护病人的健康，并降低医院、疗养院和康复设施的医疗保健费用。本项目的目标是开发一种用于检测C。很难