PNA-based microbial pathogen identification and resistance marker detection: An accurate, isothermal rapid assay based on genome-specific features.

Irina Smolina, Nancy S Miller, Maxim D Frank-Kamenetskii
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引用次数: 23

Abstract

With the rapidly growing availability of the entire genome sequences of microbial pathogens, there is unmet need for increasingly sensitive systems to monitor the gene-specific markers for diagnosis of bacteremia that enables an earlier detection of causative agent and determination of drug resistance. To address these challenges, a novel FISH-type genomic sequence-based molecular technique is proposed that can identify bacteria and simultaneously detect antibiotic resistance markers for rapid and accurate testing of pathogens. The approach is based on a synergistic combination of advanced Peptide Nucleic Acid (PNA)-based technology and signal-enhancing Rolling Circle Amplification (RCA) reaction to achieve a highly specific and sensitive assay. A specific PNA-DNA construct serves as an exceedingly selective and very effective biomarker, while RCA enhances detection sensitivity and provide with a highly multiplexed assay system. Distinct-color fluorescent decorator probes are used to identify about 20-nucleotide-long signature sequences in bacterial genomic DNA and/or key genetic markers of drug resistance in order to identify and characterize various pathogens. The technique's potential and its utility for clinical diagnostics are illustrated by identification of S. aureus with simultaneous discrimination of methicillin-sensitive (MSSA) versus methicillin-resistant (MRSA) strains. Overall these promising results hint to the adoption of PNA-based rapid sensitive detection for diagnosis of other clinically relevant organisms. Thereby, new assay enables significantly earlier administration of appropriate antimicrobial therapy and may, thus have a positive impact on the outcome of the patient.

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基于pna的微生物病原体鉴定和耐药性标记检测:基于基因组特异性特征的准确等温快速检测。
随着微生物病原体全基因组序列的迅速增加,需要越来越敏感的系统来监测用于诊断菌血症的基因特异性标记,从而能够更早地发现病原体并确定耐药性。为了解决这些挑战,提出了一种新的基于fish型基因组序列的分子技术,该技术可以识别细菌并同时检测抗生素耐药性标记,从而快速准确地检测病原体。该方法基于先进的基于肽核酸(PNA)的技术和信号增强滚动圈扩增(RCA)反应的协同结合,以实现高度特异性和敏感性的检测。特异性的PNA-DNA结构是一种极具选择性和非常有效的生物标志物,而RCA提高了检测灵敏度,并提供了一个高度多路分析系统。不同颜色的荧光修饰探针用于鉴定细菌基因组DNA中约20个核苷酸长的特征序列和/或耐药性的关键遗传标记,以鉴定和表征各种病原体。通过同时区分甲氧西林敏感(MSSA)和耐甲氧西林(MRSA)菌株来鉴定金黄色葡萄球菌,说明了该技术的潜力及其在临床诊断中的应用。总的来说,这些有希望的结果提示采用基于pna的快速敏感检测来诊断其他临床相关生物体。因此,新的检测方法使适当的抗菌素治疗能够更早地进行管理,并可能因此对患者的结果产生积极影响。
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