Urine TB diagnostic by amplicon reconstruction for PCR detection of DNA fragments

据估计，全球每年有1000万结核病病例，导致160万人死亡。结核病是世界范围内单一传染病致死的主要原因。结核病具有很高的传染性，全球遏制结核病计划指出：“…如果没有新的药物、诊断方法和有效的疫苗，我们就不会实现我们需要的…的发病率和死亡率的大幅下降。在160万人死亡中，有30万人是艾滋病毒感染者，这是一个目前的诊断经常失败的人群。在结核病最流行的发展中国家，结核病诊断依赖于临床筛查算法和痰镜检，这些都受到敏感性和特异性较低的限制。结核病检测仍然是一项重大的诊断挑战。目前的诊断方法大多是基于对痰样本中生物标志物的检测，而在儿童和艾滋病毒感染者中很难获得这一点。更容易获得的尿样已被建议作为诊断的替代患者样本。基于结核表面生物标记物LAM的尿侧流试验是可行的，但灵敏度较低。基于聚合酶链式反应的方法可能更敏感和更特异，已被提出用于检测尿液中的核酸生物标记物，但仍存在争议。尿液中结核DNA生物标记物检测的一个特别挑战是DNA降解成足够小的片段，足以通过肾脏进入尿液。尿液中的碎片特征存在三个主要挑战：1)碎片的浓度很低，2)碎片太短，无法用现有方法提取，3)生物标志物的碎片太短，无法用传统的PCR方法进行扩增。我们描述了克服这些挑战的创新方法：1)利用高梯度磁分离从大量尿液中高效地提取和浓缩片段IS6110；以及2)从短的IS6110片段实现扩增片段重构以获得全长IS6110扩增片段并进行PCR检测。有限但有希望的初步数据表明，这种方法可以应用于现有的结核病生物标记物IS6110的聚合酶链式反应。目的1对片段提取和浓缩方法进行研究。目的2研究扩增子重建方法是如何进行的，以及如何将该方法扩展到识别耐药性。这项提案旨在开发这两项技术，然后与我们在南非的合作者一起在随后的R01中在预期的患者样本中测试这些设计。

Globally, there are an estimated 10 million cases of tuberculosis (TB) each year, resulting in 1.6 million deaths. TB is the leading cause of death from a single infectious agent worldwide. TB is highly infectious and The Global Plan to Stop TB states that “… without new medicines, diagnostics and effective vaccines, we will not achieve the steep reductions in incidence and mortality that we need…” Of the 1.6 million deaths, 300,000 were HIV-infected individuals, a population where current diagnostics often fail. TB diagnostics utilized in developing countries, where TB is most prevalent, depend on clinical screening algorithms and sputum microscopy which are limited by low sensitivity and specificity. TB detection remains a significant diagnostic challenge. Current diagnostic methods are most often based on detection of biomarkers in a sputum sample which is difficult to obtain in children and HIV-infected individuals. The much more easily obtained urine sample has been suggested as an alternative patient sample for diagnosis. A urine lateral flow test for TB is available based on the TB surface biomarker LAM, but it has low sensitivity. PCR-based approaches, which potentially are much more sensitive and specific, have been proposed for detecting nucleic acid biomarkers in urine but remain controversial. A particular challenge for TB DNA biomarker testing in urine is DNA degradation into fragments small enough to pass through the kidney into the urine. The characteristics of fragmentation in urine presents three main challenges: 1) fragments are present in very low concentrations, 2) fragments present are too short for extraction by available methods, and 3) fragments of biomarkers are too short for amplification by traditional PCR methods. We describe innovative methods to overcome these challenges to 1) achieve efficient extraction and concentration of fragmented IS6110 from large volumes of urine using high gradient magnetic separation, and 2) a method of achieving amplicon reconstruction from short IS6110 fragment to make full-length IS6110 amplicons and enable PCR detection. Limited, but promising, preliminary data suggest this approach can be applied to existing PCR reactions for the TB biomarker IS6110. Aim 1 studies are proposed to characterize the fragment extraction and concentration approach. Aim 2 examines how the amplicon reconstruction approach performs and how this method can be extended to identify drug resistance. This proposal aims to develop these two technologies before testing these designs in prospective patient samples in a subsequent R01 with our collaborators in South Africa.