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.

全球每年估计有 1000 万结核病 (TB) 病例，导致 160 万人死亡，结核病具有传染性，全球控制结核病计划指出。 “……如果没有新药、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大幅降低……”在 160 万人死亡中，有 30 万人是艾滋病毒感染者，在结核病最流行的发展中国家，目前的结核病诊断方法常常失败，而结核病检测的灵敏度和特异性有限，这仍然是一个重大的挑战。最常见的是基于痰样本中生物标志物的检测，这在儿童和艾滋病毒感染者中很难获得，而更容易获得的尿液样本被建议作为诊断的替代患者样本。 TB 可以基于 TB 表面生物标志物 LAM 进行检测，但其灵敏度较低，而这种方法可能具有更高的敏感性和特异性，已被提议用于检测尿液中的核酸生物标志物，但仍存在争议。尿液中的 DNA 生物标志物测试是将 DNA 降解为足够小的片段，以便通过肾脏进入尿液。尿液中的片段化特征提出了三个主要挑战：1) 片段的浓度非常低，2) 片段的长度太短，无法检测到。通过现有方法提取， 3) 生物标志物片段太短，无法通过传统 PCR 方法进行扩增，我们描述了克服这些挑战的创新方法，以 1) 使用高梯度磁力分离从大量尿液中实现碎片化 IS6110 的有效提取和浓缩，以及 2)从短 IS6110 片段实现扩增子重建以制备全长 IS6110 扩增子并实现 PCR 检测的方法有限但有前景的初步数据表明该方法可应用于现有的结核病 PCR 反应。目标 1 研究旨在描述片段提取和浓缩方法的特征，以及如何扩展该方法以识别耐药性。该提案旨在在测试这些设计之前开发这两种技术。我们与南非的合作者在随后的 R01 中对前瞻性患者样本进行了研究。