用于结核分枝杆菌高效耐药性检测的高通量纳米孔靶向测序技术

Chen Tang, Lianpeng Wu, Machao Li, Jianyi Dai, Ye Shi, Qiongdan Wang, Feng Xu, Laibao Zheng, Xingxing Xiao, Junwen Cai, Yanjun Zhang, Yuting Yang, Xiaoqun Zheng, Guangxin Xiang
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引用次数: 0

摘要

耐药性结核病(TB),尤其是耐多药结核病(MDR-TB)和广泛耐药结核病(XDR-TB),是亟待解决的临床问题和公共卫生挑战之一。基于培养基的表型药敏试验(pDST)耗时较长,而基于 PCR 的检测仅限于热点突变。在本研究中,我们开发并验证了一种基于高通量纳米孔测序技术结合多重 PCR 的便捷高效方法,即纳米孔靶向测序(NTS),可同时对结核分枝杆菌(MTB)中与抗生素耐药性相关的 18 个基因进行测序。对 NTS 的分析性能进行了评估,并收集了 99 份临床样本以评估其临床性能。此外,与 pDST 和 Xpert MTB/RIF 检测法相比,NTS 提供了更多的耐药性信息,涵盖了 14 种抗结核药物,并发现了 20 例耐药 MTB 临床病例。这些耐药病例的基因突变都是通过 Sanger 测序验证的。我们的结核病耐药性检测方法具有无需培养、高效、高通量和高准确性等优点,对临床病人管理和结核病感染控制非常有帮助。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis
Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.
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