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

全球每年估计有1000万例结核病病例，造成160万人死亡。结核病是世界范围内单一传染性病原体导致死亡的主要原因。结核病具有高度传染性，《遏制结核病全球计划》指出，“ .如果没有新的药物、诊断方法和有效的疫苗，我们就无法实现我们所需要的发病率和死亡率的急剧下降。”在160万死亡者中，有30万人是艾滋病毒感染者，而目前的诊断方法往往无法对这一人群进行诊断。在结核病最流行的发展中国家使用的结核病诊断依赖于临床筛查算法和痰显微镜，而这些算法受到低灵敏度和特异性的限制。结核病检测仍然是一项重大的诊断挑战。目前的诊断方法通常是基于检测痰样本中的生物标志物，这在儿童和艾滋病毒感染者中很难获得。更容易获得的尿液样本已被建议作为诊断的替代患者样本。基于结核表面生物标志物LAM的结核尿侧流试验是可用的，但它的灵敏度较低。基于pcr的方法可能更加敏感和特异性，已被提出用于检测尿液中的核酸生物标志物，但仍存在争议。在尿液中进行结核病DNA生物标志物测试的一个特别挑战是DNA降解成足够小的片段，可以通过肾脏进入尿液。尿液中碎片的特征提出了三个主要挑战：1)碎片的浓度很低，2)存在的碎片太短，无法用现有方法提取，3)生物标志物的片段太短，无法用传统的PCR方法扩增。我们描述了克服这些挑战的创新方法，1)利用高梯度磁分离技术从大量尿液中高效提取和浓缩碎片化的IS6110，以及2)从短IS6110片段中实现扩增子重建以制备全长IS6110扩增子并进行PCR检测的方法。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志物IS6110的PCR反应。目的1研究提出了表征碎片提取和浓缩方法。目的2检查扩增子重建方法如何执行，以及如何将该方法扩展到识别耐药性。该提案旨在开发这两种技术，然后与我们在南非的合作者在随后的R01中对这些设计在前瞻性患者样本中进行测试。

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.