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.

新出现的抗微生物药物耐药性增加了治疗细菌性败血症等关键疾病的复杂性。诊断特定耐药性的方法往往是快速或敏感的，但不能两者兼而有之。如果序列特异性单分子分析等检测方法适用于类似于传统临床情况下产生的较小目标，则可以解决这一问题。在这项工作中，我们证明了可以通过使用生物素/链霉亲和素磁珠系统固定 DNA、用 SYBR Gold 标记并在探针保留的同时冲洗掉多余的部分来创建具有悬垂单链 25 bp 探针序列的 120 bp 双链多核苷酸多个荧光团。然后，这些带有多个荧光团的探针可用于以序列特异性方式标记细菌质粒目标。这些探针能够检测含有部分抗生素抗性基因序列的 1 pM 质粒样品。该系统显示了改进小核酸片段的捕获和荧光标记的可能性，在保持快速处理时间的同时为临床相关样品产生较低的检测限。