Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL.
Department of Chemistry, College of Sciences, University of Central Florida, Orlando, FL.
Clin Chem. 2019 Feb;65(2):333-341. doi: 10.1373/clinchem.2018.295212. Epub 2018 Dec 6.
Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from (). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories.
In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTM). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (, , , , , and ) and .
Using the fluorescent BiDz-NTM assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye.
The BiDz-NTM assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.
非结核分枝杆菌（NTM）是一种日益严重的威胁，尤其是对患有肺部合并症的患者。目前的即时检测诊断未能充分识别和区分 NTM 物种与（）。基于培养和分子的明确检测可能需要数周至数月的时间，并且需要将样本送到专门的诊断实验室。
在这项概念验证研究中，我们开发了一种基于聚合酶链反应（PCR）扩增 16S 核糖体 RNA（rRNA）基因的检测方法，使用通用分枝杆菌引物，并使用一组二元脱氧核酶（BiDz）传感器对扩增片段进行检测，以实现 NTM 的种水平鉴定（BiDz-NTM）。每个 BiDz 传感器由 2 个 RNA 切割脱氧核酶的亚基组成，只有在存在互补靶序列的情况下，才能形成有活性的脱氧核酶催化核心。靶标激活的 BiDz 催化报告底物的切割，从而根据所用的底物触发荧光或比色（肉眼观察）信号。该小组包括用于区分 6 种临床相关 NTM 物种（，，，，，和）和的 BiDz 传感器。
使用荧光 BiDz-NTM 检测，我们成功地鉴定了 38 株临床分离株的物种。此外，用可视 BiDz 传感器测试了一部分菌株，为 NTM 的种型鉴定提供了通过肉眼进行的概念验证。
BiDz-NTM 检测是一种快速鉴定 NTM 物种的新平台。该方法具有高度特异性，比目前的工具快得多，并且易于适应医院或临床实验室的现场诊断实验室。
