A fully integrated assay and platform for detecting Clostridium difficile

描述（由申请人提供）：有害细菌感染的抗生素治疗可破坏正常的结肠菌群，增加患者对艰难梭菌相关疾病（CDAD）的易感性。艰难梭菌的致病菌株通常产生两种大的梭菌毒素，A （TcdA）和B (TcdB)，负责CDAD。TcdC基因是TcdA和TcdB基因的负调控因子，而最具毒性的艰难梭菌BI/NAP1在该基因上有一个单点突变，导致过早停止密码子。在这些菌株中缺乏功能性的TcdC蛋白导致TcdA和TcdB毒素的过度表达。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艰难梭菌感染患者，需要对CDAD，特别是BI/NAP1株具有鲁棒性和特异性的简单检测方法。目前广泛使用的EIA测试快速但缺乏灵敏度，而更敏感的测试（细胞毒性试验或艰难梭菌培养）需要一天或更长时间才能完成。显然，还需要一种可靠、灵敏和快速的方法来检测环境污染。一种能够处理和检测艰难梭菌孢子的灵敏检测方法将在很大程度上保护患者的健康，并降低CDAD在医院、疗养院和康复机构传播的风险。大多数病例的缓解是由于再次感染。去污是非常困难的，目前没有可靠和敏感的核查方法支持。在这个第一阶段，我们建议开发一种简单的检测系统，用于检测TcdA基因，TcdB基因和TcdC点突变，使用专有的解旋酶依赖扩增（HAD”）技术。HDA反应将检测TcdA是否存在和携带艰难梭菌，TcdC D117突变是否存在高毒力艰难梭菌。我们将开发一种即时护理（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稳健且简单，避免了洗涤步骤、洗脱缓冲液或离心的需要。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独立的功能盒中，一次使用后是一次性的，也可以与其他模块连接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，以前曾与HDA分析方法一起使用。

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.