■准确检测和鉴定病原体及其相关的耐药机制是在耐碳青霉烯类肠杆菌(CRE)的管理中实施精准医学的必要前提。在各种抵抗机制中,KPC碳青霉烯酶的生产是世界上最普遍的。因此,本研究旨在开发一种简便、精确的blaKPC基因核酸检测平台。
■我们研究方法的初始阶段涉及开发CRISPR/Cas12a检测框架,这是通过设计靶向blaKPC基因的高度特异性单向导RNA(sgRNA)来实现的。为了提高系统的灵敏度,我们结合了三种不同的扩增技术-聚合酶链反应(PCR),环介导等温扩增(LAMP),和重组酶聚合酶扩增(RPA)-进入CRISPR/Cas12a框架。随后,我们对这三种扩增方法与CRISPR/Cas12a系统联合使用时的敏感性和特异性进行了比较分析.此外,我们通过评估80种不同临床分离株的荧光读数,评估了这些方法学的临床适用性.此外,我们采用侧流测定技术来提供结果的直观表示,促进即时测试。
■在对三种方法的敏感性和特异性进行比较分析之后,我们确定RPA-Cas12a方法是最佳检测技术.我们的发现表明,RPA-Cas12a平台的检测限(LoD)对于质粒DNA为1aM(〜1拷贝/µL),对于基因组DNA为5×109.3fg/µL。此外,在对80株临床分离株进行验证后,该平台的敏感性和特异性均达到100%.
■这些发现表明,开发的RPA-Cas12a平台代表了具有成本效益的有前途的工具,方便,并准确检测blaKPC基因。
UNASSIGNED: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the blaKPC gene.
UNASSIGNED: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the blaKPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing.
UNASSIGNED: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates.
UNASSIGNED: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the blaKPC gene.