Development of a method for visual detection of Mycobacterium tuberculosis complex

项目摘要 结核病，一种由结核分枝杆菌引起的传染病 复杂(MTC)，仍然是全球主要公共卫生问题之一，有900万新的 每年约有150万人死亡。此外，抗生素的不适当使用在 对药物敏感的结核病患者进行化疗会产生抗药性， 使结核病护理和控制复杂化。约95%的致命病例发生在资源有限的高负担地区 缺乏诊断能力的国家。未诊断或诊断不正确 患者会发展成更严重的疾病，并仍然是更广泛公众的感染源。在… 同时，在低资源设置下的高效护理点(POC)结核病诊断可以改进 通过促进更有效的治疗、降低患者辍学率和 限制感染的传播。这种形式的检测缺乏药物敏感性检测 (DST)。因此，新的诊断工具足够简单，可以在现场使用，并且 仍然需要能够正确诊断结核病并识别抗药性。这样做的目的是 高风险、高回报的勘探项目是利用最近在 DNA纳米技术和分子传感显著提高诊断能力 结核病。我们建议开发与PoC兼容的高选择性传感器，基于以下级联 具有视觉信号读数的脱氧核酶用于检测活动性肺结核和检测 导致多药耐药(MDR)的突变。可视脱氧核酶级联传感器将 能够在正常就诊期间产生高度可靠的结果，并且 在不需要昂贵设备的情况下，能够检测到不到104个MTC细胞。 我们将设计针对mtc rRNA和含有point的基因片段的级联传感器。 对最有效的一线抗结核药物利福平和异烟肼产生抗药性的突变。 我们将使用分离的细菌核酸、整个细菌细胞和 被细菌污染的人类唾液样本。我们会将化验结果与抗酸涂片进行比较。 显微镜和基于培养的测试，目前用于结核病检测和DST在 资源受限设置。这里提出的技术在结核病的诊断方面很有前途，因为 以及其他病原体，特别是在资源有限的高负担国家，这可能 使全球医疗保健发生革命性变化。

Project Summary Tuberculosis (TB), an infectious disease caused by species of Mycobacterium tuberculosis complex (MTC), remains one of the major public health problems worldwide, with nine million new cases and about 1.5 million deaths each year. In addition, improper use of antibiotics in chemotherapy of drug-susceptible TB patients results in anti-TB drug resistance, which complicates TB care and control. About 95% of lethal cases occur in resource-limited high-burden countries, where there is a lack of diagnostic capacity. Undiagnosed or improperly diagnosed patients develop more severe disease and remain a source of infection for the wider public. At the same time, efficient point-of-care (POC) TB diagnostics in low resource settings can improve health outcomes by facilitating more effective treatment, reducing the patient dropout rate and limiting spread of the infection. The tests of such format are lacking for drug susceptibility testing (DST) of TB. Therefore, new diagnostic tools that are simple enough to be used “in the field” and still be able to correctly diagnose for TB and identify drug-resistance are needed. The goal of this high-risk, high-reward exploratory project is to take advantage of the recent advancements in DNA nanotechnology and molecular sensing to significantly improve diagnostics capabilities for TB. We propose to develop POC-compatible highly selective sensors based on cascades of deoxyribozymes with visual signal readout for detection of active pulmonary TB and testing for mutations conferring multidrug resistance (MDR). Visual deoxyribozyme cascade sensors will be able to produce highly reliable results within the period of usual doctor's visit, and be able to detect less than 104 cells of MTC species without the need for expensive equipment. We will design cascade sensors targeting MTC rRNA and fragments of the genes containing point mutations conferring resistance to the most potent first-line anti-TB drugs rifampicin and isoniazid. We will optimize the assay using isolated bacterial nucleic acids, whole bacterial cells and bacteria-spiked human sputum samples. We will compare the assay with acid fast smear microscopy and culture-based tests, which are currently used for TB detection and DST in the resource-limited settings. The technology proposed here is promising for diagnostics of TB, as well as other pathogens, particularly in resource-constrained high-burden countries, which may revolutionize healthcare worldwide.