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

在全球范围内，每年估计有1 000万例结核病病例，造成160万人死亡。结核病是全球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，《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，我们将无法实现我们所需要的发病率和死亡率的急剧下降.”在160万例死亡中，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，结核病诊断依赖于临床筛查算法和痰液显微镜检查，这受到灵敏度和特异性低的限制。结核病检测仍然是一个重大的诊断挑战。目前的诊断方法通常基于痰液样本中的生物标志物检测，这在儿童和HIV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TB表面生物标志物LAM，可进行TB的尿液侧流试验，但其灵敏度较低。基于PCR的方法，这可能是更敏感和特异性，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。尿液中TB DNA生物标志物检测的一个特殊挑战是DNA降解成足够小的片段，以通过肾脏进入尿液。尿液中片段化的特征提出了三个主要挑战：1）片段以非常低的浓度存在，2）存在的片段太短而无法通过可用方法提取，以及3）生物标志物的片段太短而无法通过传统PCR方法扩增。我们描述了克服这些挑战的创新方法，以1）使用高梯度磁分离实现从大量尿液中有效提取和浓缩片段化的IS6110，以及2）实现从短IS6110片段重建扩增子以制备全长IS6110扩增子并实现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TB生物标志物IS6110的现有PCR反应。目的1研究提出了表征片段提取和浓缩方法。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鉴定耐药性。该提案旨在与我们在南非的合作者在随后的R01中在前瞻性患者样本中测试这些设计之前开发这两种技术。

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.