Abstract

Bacterial beta-lactamases and carbapenemases confer resistance to beta-lactam antibiotics, including penicillins, cephalosporins, carbapenems, and monobactams. Their wide distribution among the bacteria that cause infectious diseases in humans and animals represent a global threat. We have developed a biochip with colorimetric detection based on horseradish peroxidase for the simultaneous identification of genes for all clinically relevant class A beta-lactamases and carbapenemases of classes A, B, and D, including 25 single substitutions in the nucleotide sequence encoding the key amino acid substitutions in class A beta-lactamases. The conditions for allele-specific hybridization of biotin-labeled target DNA with oligonucleotide probes immobilized on the surface of the biochip have been optimized. A method of multiplex amplification of all of the studied genes in one reaction with the simultaneous incorporation of biotin was developed to obtain the target DNA. The biochip was validated with mixtures of the beta-lactamase and carbapenemase genes, as well as 68 DNA samples isolated from clinical strains of gram-negative bacteria. The total DNA sample analysis time was ~4 h. A high specificity of the identification of genes in mixtures was demonstrated, which can be used in the study of multidrug-resistant bacteria.

中文翻译：

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同时识别β-内酰胺酶和碳青霉烯酶基因赋予细菌对β-内酰胺抗生素耐药性的生物芯片。

摘要

细菌β-内酰胺酶和碳青霉烯酶赋予了对β-内酰胺抗生素（包括青霉素，头孢菌素，碳青霉烯和单杆菌素）的抗性。它们在引起人类和动物传染病的细菌中广泛分布，这构成了全球性威胁。我们已经开发了一种基于辣根过氧化物酶的比色检测生物芯片，用于同时鉴定所有临床相关的A类β-内酰胺酶和A，B和D类碳青霉烯酶的基因，包括编码关键氨基酸的核苷酸序列中的25个单取代A类β-内酰胺酶中的酸取代。生物素标记的靶DNA与固定在生物芯片表面的寡核苷酸探针的等位基因特异性杂交的条件已得到优化。开发了在一个反应​​中同时掺入生物素来多重扩增所有研究基因的方法，以获得目标DNA。用β-内酰胺酶和碳青霉烯酶基因的混合物以及从革兰氏阴性细菌临床菌株中分离出的68个DNA样品对生物芯片进行了验证。DNA样品的总分析时间为〜4 h。证明了混合物中基因鉴定的高度特异性，可用于耐多药细菌的研究。