PNA-based microbial pathogen identification and resistance marker detection: An accurate, isothermal rapid assay based on genome-specific features.
PNA-based microbial pathogen identification and resistance marker detection: An accurate, isothermal rapid assay based on genome-specific features.
复制标题
DOI：
10.4161/adna.1.2.13256
复制
发表时间：
2010-10-01
期刊：
影响因子：
--
通讯作者：
Frank-Kamenetskii, Maxim D
中科院分区：
文献类型：
--
作者：
Smolina, Irina;Miller, Nancy S;Frank-Kamenetskii, Maxim D
随着微生物病原体的全基因组序列的快速增长的可用性，对于监测用于诊断菌血症的基因特异性标志物的日益敏感的系统存在未满足的需求，所述系统使得能够更早地检测病原体和确定耐药性。为了应对这些挑战，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，以快速准确地检测病原体。该方法基于先进的肽核酸（PNA）技术和信号增强滚环扩增（RCA）反应的协同组合，以实现高度特异性和灵敏度的测定。特异性PNA-DNA构建体作为一种高度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重的测定系统。不同颜色的荧光装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。该技术的潜力和临床诊断的实用性说明了通过鉴定的S。金黄色葡萄球菌，同时区分甲氧西林敏感（MSSA）与甲氧西林耐药（MRSA）菌株。总的来说，这些有希望的结果暗示了采用基于PNA的快速灵敏检测来诊断其他临床相关的生物体。随着微生物病原体全基因组序列的快速增长，对监测用于诊断菌血症的基因特异性标志物的日益敏感的系统的需求未得到满足，所述系统使得能够更早地检测病原体和确定耐药性。为了应对这些挑战，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，以快速准确地检测病原体。该方法基于先进的肽核酸（PNA）技术和信号增强滚环扩增（RCA）反应的协同组合，以实现高度特异性和灵敏度的测定。特异性PNA-DNA构建体作为一种高度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重的测定系统。不同颜色的荧光装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。该技术的潜力和临床诊断的实用性说明了通过鉴定的S。金黄色葡萄球菌，同时区分甲氧西林敏感（MSSA）与甲氧西林耐药（MRSA）菌株。总的来说，这些有希望的结果暗示了采用基于PNA的快速灵敏检测来诊断其他临床相关的生物体。因此，新的测定能够显著更早地施用适当的抗微生物疗法，并且因此可能对患者的结果具有积极影响。
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.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.