Transrenal DNA for point-of-care diagnosis of TB

摘要 结核分枝杆菌(Mtb)感染了世界三分之一的人口，每年导致1000万人患有结核病， 每年造成160万人死亡。虽然结核病的有效治疗方法已经存在了多年 50年来，控制该病的一个主要障碍仍然是无法在资源不足的情况下可靠地诊断疾病 结核病流行环境。大多数临床测试需要足够的痰样本(来自肺部的粘液)， 在没有咳嗽的患者、儿童和艾滋病毒感染者中可能很难获得。尿液提供了一种 这是一种很有吸引力的现场结核病诊断方法，因为它丰富，收集简单，而收集不是 涉及到接触传染性气雾剂。尿液中存在的经肾DNA(TrDNA)提供了一个特定的靶点 可放大以实现高灵敏度。尿液结核聚合酶链式反应检测方法的建立及发展 作为一种护理点检测，可能会对结核病诊断产生巨大影响。 目的1.改进超短聚合酶链式反应和多重扩增检测结核分枝杆菌特异性trDNA的方法。假设： 扩增较短的片段和/或靶向多个独特的片段将能够检测到低- 浓缩样品，在保持特异度的同时提高灵敏度。 目的2.测定改良结核分枝杆菌特异性trDNA检测尿液的敏感性和特异性 样本来自结核病流行环境中的成人和儿童。假设：MTB trDNA存在于可检测到的 成人和儿童肺结核病患者的尿量与艾滋病毒状况无关，我们的 与以往的结核分枝杆菌trDNA研究相比，杂交序列捕捉法可以提高临床敏感性。

ABSTRACT M. tuberculosis (Mtb) infects one-third of the world’s population, causes TB disease in 10 million people per year, and is responsible for 1.6 million deaths annually. While effective treatments for TB have been available for over 50 years, a major barrier to its control has remained the inability to reliably diagnose disease in low-resource TB-endemic settings. Most clinical tests require an adequate sample of sputum (mucus from the lungs) which may be difficult to obtain in patients without a cough, in children, and in HIV-infected individuals. Urine offers an attractive method for point-of-care TB diagnostics since it is abundant, simple to collect, and collection does not involve exposure to infectious aerosols. Transrenal DNA (trDNA) present in the urine offers a specific target that can be amplified for high sensitivity. The development of a PCR-based test of TB from urine, and its development as a point-of-care test, could have enormous impact on TB diagnostics. Aim 1. To improve detection of Mtb-specific trDNA using ultrashort PCR and multiplexing. Hypothesis: Amplification of shorter fragments and/or targeting multiple unique fragments will enable detection of low- concentrations samples, increasing sensitivity while maintaining specificity. Aim 2. To determine sensitivity and specificity of improved Mtb-specific trDNA detection in urine samples from adults and children in a TB-endemic setting. Hypothesis: Mtb trDNA is present in detectable quantities in urine from both adults and children with pulmonary TB regardless of HIV status, and our hybridization sequence-capture method can increase clinical sensitivity compared to previous TB trDNA studies.