Tuberculosis Second Line Drug Susceptibility Chip

描述（由申请人提供）：本提案涉及RFA-AI-10-003“生物防御合作伙伴关系”，并将学术研究人员、行业、MDR TB临床医生、BSL 3实验室和现场研究中心聚集在一起，为多重和广泛耐药结核病开发更快、定量的二线药物敏感性试验（DST）。耐药结核病（C类威胁）的出现导致了一种高度传播且在某些情况下无法治愈的疾病的不祥局面。几个Tb DST平台正在使用，但都有很大的限制，特别是周转时间。我们认为，我们必须坚持基于表型培养的方法，因为直接分子检测对于二线药物仍然不明确，但我们已经证明，可以通过更早的3天定量PCR读数来改善现有的液体方法，该读数测量药物环境中的DNA扩增。通过使用高通量小体积微量滴定板，我们的方法可以产生准确的快速定性结果和类似于MIC的定量“抑制指数”-临床上需要但很少用于Tb的测量。由于MDR/XDR Tb对实验室人员的生物安全性至关重要，因此我们将该测定转化为新颖设计、封闭系统、一次性、具有qPCR能力的微流控芯片。这种微流控室可以精确地计量每个孔中的杆菌数量，最大限度地减少一个重现性差的区域，并且还可以通过qPCR以外的其他参数原位测量生长，包括新发明的DNA检测技术，我们称之为风车。因此，qPCR检测开发和芯片项目将受益于彼此的成功，但并不依赖于彼此的成功。我们的目标是为二线药物提供一个改进的DST平台，以更快地提供信息，更好地治疗和减少MDR/XDR Tb的传播。MDR和XDR Tb的药物敏感性测试是缓慢的，并且充满了技术困难。该提案将开发一种快速、定量的基于PCR的诊断方法，以在3天内以微孔板形式检测Tb对二线药物的敏感性或耐药性。该分析将适用于封闭系统的一次性芯片，以提高实验室人员的生物安全性，并允许通过我们新的“风车”方法进行DNA定量。这些结果将立即被MDR Tb临床医生用于更好地治疗和减少MDR/XDR Tb的传播。

DESCRIPTION (provided by applicant): This proposal addresses RFA-AI-10-003 "Partnerships for Biodefense" and brings together academic researchers, industry, MDR TB clinicians, BSL3 Labs, and field sites to develop a faster, quantitative, second line drug susceptibility test (DST) for multiple and extensively drug-resistant tuberculosis. The emergence of drug resistant TB, a Category C threat, has led the ominous situation of a highly transmissible and in some instances untreatable disease. Several Tb DST platforms are in use yet all have significant constraints, particularly turnaround time. We feel that we must persevere with a phenotypic culture based method, since direct molecular testing will remain poorly defined for second line drugs, but we have shown that can improve existing liquid methods with an earlier 3 day quantitative PCR-based readout that measures DNA amplification in the setting of drug. By using high-throughput small-volume microtiter plates, our method can yield both an accurate rapid qualitative result and a quantitative "inhibition index" akin to the MIC - a measurement that is clinically needed but rarely available for Tb. Because biosafety of MDR/XDR Tb for laboratory personnel is of utmost importance, we will then transform this assay into a novel-design, closed- system, disposable, qPCR-capable microfluidic chip. This microfluidic chamber can accurately meter the number of bacilli fed into each well, minimizing one area of poor reproducibility, and can also measure growth in situ by other parameters beyond qPCR, including a newly-invented DNA detection technique we term pinwheel. Thus the qPCR assay development and chip project will benefit from but are not tied to each other's success. The goal is an improved DST platform for second line drugs that yields faster information to better treat and decrease transmission of MDR/XDR Tb. NARRATIVE Drug susceptibility testing for MDR and XDR Tb is slow and fraught with technical difficulties. This proposal will develop a rapid, quantitative PCR-based diagnostic to detect susceptibility or resistance of Tb to second line drugs in a microplate format within 3 days. The assay will be adapted to a closed-system disposable chip to enhance biosafety for lab personnel and permit DNA quantification by our new 'pinwheel' approach. The results will be immediately usable by MDR Tb clinicians to better treat and decrease transmission of MDR/XDR Tb.