Multilabel hybridization probes for sequence-specific detection of sepsis-related drug resistance genes in plasmids.
Multilabel hybridization probes for sequence-specific detection of sepsis-related drug resistance genes in plasmids.
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DOI：
10.1016/j.talo.2021.100034
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发表时间：
2021-08
期刊：
影响因子：
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通讯作者：
R. Hanson;Elaine Lazalde;R. Knob;David H. Harris;Yesman Akuoko;J. B. Nielsen;A. Woolley
中科院分区：
文献类型：
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作者：
R. Hanson;Elaine Lazalde;R. Knob;David H. Harris;Yesman Akuoko;J. B. Nielsen;A. Woolley
新出现的抗微生物药物耐药性增加了治疗细菌诱导的脓毒症等关键疾病的复杂性。诊断特异性耐药性的方法往往是快速或敏感的，但不是两者兼而有之。像序列特异性单分子分析这样的检测方法可以解决这个问题，如果它们可以适应于类似于传统临床情况下产生的较小靶标。在这项工作中，我们证明了一个120 bp的双链多核苷酸与突出的单链25 bp的探针序列可以通过固定DNA与生物素/链霉亲和素磁珠系统，标记SYBR金，并冲洗掉多余的，而探针保留多个荧光团。然后，这些具有多个荧光团的探针可以用于以序列特异性方式标记细菌质粒靶标。这些探针能够检测含有部分抗生素耐药基因序列的1 pM质粒样品。该系统显示了改善小核酸片段的捕获和荧光标记的可能性，从而在保持快速处理时间的同时产生临床相关样品的检测下限。
Emerging antimicrobial drug resistance is increasing the complexity involved in treating critical conditions such as bacterial induced sepsis. Methods for diagnosing specific drug resistance tend to be rapid or sensitive, but not both. Detection methods like sequence-specific single-molecule analysis could address this concern if they could be adapted to work on smaller targets similar to those produced in traditional clinical situations. In this work we demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be created by immobilizing DNA with a biotin/streptavidin magnetic bead system, labeling with SYBR Gold, and rinsing the excess away while the probe retains multiple fluorophores. These probes with multiple fluorophores can then be used to label a bacterial plasmid target in a sequence-specific manner. These probes enabled the detection of 1 pM plasmid samples containing a portion of an antibiotic resistance gene sequence. This system shows the possibility of improving capture and fluorescence labeling of small nucleic acid fragments, generating lower limits of detection for clinically relevant samples while maintaining rapid processing times.