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

通过扩增子重建进行 DNA 片段 PCR 检测诊断尿结核

• 批准号: 10385847
• 负责人: Frederick R Haselton
• 金额: $ 19.43万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-06 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10385847
• 关键词: Accounting; Algorithms; Beds; Biological Markers; Blood; Cause of Death; Cessation of life; Characteristics; Child; Clinical; Collaborations; Complex; Country; DNA; Data; Deoxyribonuclease I; Detection; Developing Countries; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic Sensitivity; Diagnostic tests; Drug resistance; Future; HIV; Human; Incidence; Individual; Infection; Infectious Agent; Kidney; Length; Letters; Magnetism; Measurement; Medicine; Methodology; Methods; Microscopy; Modification; Nucleic Acids; Nucleotides; Patients; Performance; Persons; Polymerase Chain Reaction; Population; Publishing; Reaction; Reagent; SNP genotyping; Sampling; Scheme; Sensitivity and Specificity; Site; South Africa; Specificity; Sputum; Stains; Surface; System; Technology; Testing; Time; Tuberculosis; Urine; Vaccines; antigen test; base; cancer biomarkers; design; detection limit; fetal; improved; innovation; interest; lateral flow assay; magnetic beads; mortality; prospective; reconstruction; screening; synthetic construct; tuberculosis diagnostics; urinary

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.

在全球范围内，估计每年有1000万例结核病（TB），导致160万例死亡。结核病是全世界单个传染毒剂死亡的主要原因。结核病具有高度感染力，全球计划阻止结核病指出：“……如果没有新药物，诊断和有效的疫苗，我们将无法实现我们需要的发病率和死亡率的减少……”在160万人死亡中，有30万人是艾滋病毒感染者，是艾滋病毒感染者，是当前诊断的人群。结核病最普遍的发展中国家使用的结核病诊断取决于临床筛查算法和痰液显微镜，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个重大的诊断挑战。当前的诊断方法通常是基于对痰样品中生物标志物的检测，而生物标志物在儿童和HIV感染的个体中很难获得。已建议更容易获得的尿液样本作为诊断诊断的替代患者样本。根据结核表面生物标志物LAM，可为结核病的尿液横向流程测试，但其灵敏度较低。已经提出了基于PCR的方法更敏感和特异性，用于检测尿液中的核酸生物标志物，但仍存在争议。 TB DNA生物标志物在尿液中的一个特殊挑战是DNA降解成小的片段，足以通过肾脏进入尿液。尿液中碎片化的特征提出了三个主要挑战：1）碎片以非常低的浓度，2）存在的碎片太短，无法通过可用方法提取，而3）生物标志物的碎片太短了，无法通过传统的PCR方法扩增。我们描述了克服这些挑战的创新方法。1）使用高梯度磁分离从大量的尿液中实现有效的提取和块状IS6110，以及2）一种从短IS6110碎片中实现放大器重建的方法，以使全长IS6110 IS6110 iS6110 amplifier和启用PCR检测。有限但承诺的初步数据表明，这种方法可以应用于结核病生物标志物IS6110的现有PCR反应。提出了AIM 1研究以表征碎片提取和浓度方法。 AIM 2检查放大器重建方法的执行方式以及如何扩展该方法以识别耐药性。该提案旨在在随后的R01中与我们在南非的合作者在后续的R01中测试这些设计之前，在对这些设计进行测试之前开发这两种技术。