Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America.
Flemish Institute for Technological Research, VITO, Mol, Belgium.
PLoS One. 2019 Feb 7;14(2):e0212064. doi: 10.1371/journal.pone.0212064. eCollection 2019.
In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (<5%) of drug-resistant Mtb was possible.
在结核分枝杆菌（Mtb）中，单核苷酸多态性（SNP）的检测无论是在诊断方面，还是在流行病学研究中都非常重要。在诊断方面，由于耐药性主要是由于多个药物靶点的 SNP 获得引起的，因此 SNP 的检测对于药物耐药性的检测至关重要；在流行病学研究中，通过 SNP 识别进行菌株分型。为了提供对临床相关耐药谱和菌株类型的必要覆盖，基于核酸的测量技术必须能够检测大量潜在的 SNP。由于 Mtb 问题在许多资源匮乏的国家非常紧迫，需要低成本的即时护理生物传感器，因此这是一个具有挑战性的技术问题。本文提出了一个概念验证，我们选择简单的 DNA-DNA 杂交作为传感原理，因为它可以转移到现有的低成本硬件平台上，并且我们推动了它的多重化边界。通过使用自定义设计的探针集和物理化学驱动的数据分析，可以同时检测与一线和二线药物耐药性以及 Mtb 菌株分型相关的 SNP 的存在。我们已经证明了它可用于从一组系统发育多样化的临床菌株中鉴定药物耐药性和菌株类型。此外，还可以可靠地检测出少数（<5%）耐药 Mtb 的存在。
