Isothermal amplification reactions represent an important and exciting approach to achieve widespread, low cost, and easily implemented molecular diagnostics. This work presents a modified recombinase polymerase amplification (RPA) reaction, which can be directly coupled to a simple electrochemical measurement to ultimately allow development of a nucleic acid-based assay for antibiotic resistance genes. It is shown that use of reagents from a standard RPA reaction kit allows incorporation of horse radish peroxidase-labeled thymine nucleotides into amplified DNA strands, which can be detected via an amperometric signal readout for detection of important gene sequences. The assay is exemplified through detection of fragments of the oxacillin resistance gene in Escherichia coli cells bearing a drug resistance plasmid, achieving a potential limit of detection of 319 cfus/mL and an unoptimized time to result of 60 min. This work serves as a suitable demonstration of the potential for a system to deliver detection of key drug resistance genes at clinically relevant levels.

中文翻译：

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使用直接标记固相等温扩增法对苯唑西林耐药性进行电化学检测

等温扩增反应是实现广泛、低成本且易于实施的分子诊断的重要且令人兴奋的方法。这项工作提出了一种改良的重组酶聚合酶扩增 (RPA) 反应，它可以直接与简单的电化学测量相结合，最终允许开发基于核酸的抗生素抗性基因检测。结果表明，使用标准 RPA 反应试剂盒中的试剂可以将辣根过氧化物酶标记的胸腺嘧啶核苷酸掺入扩增的 DNA 链中，这可以通过安培信号读数进行检测，以检测重要的基因序列。该测定通过检测大肠杆菌中苯唑西林抗性基因的片段来举例说明带有抗药性质粒的细胞，达到 319 cfus/mL 的潜在检测限和 60 分钟的未优化结果时间。这项工作适当地证明了系统在临床相关水平上检测关键耐药基因的潜力。