State Key Laboratory for Manufacturing Systems Engineering, School of Instrument Science and Techonology, Xi'an Jiaotong University, Xi'an 710054, China.
Biosensors (Basel). 2024 Nov 13;14(11):549. doi: 10.3390/bios14110549.
Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the and genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.
簇状规律间隔短回文重复序列 (CRISPR) 分子诊断技术是最可靠的传染病诊断工具之一，因为它具有反应时间短、灵敏度高和特异性好的特点。然而，与荧光聚合酶链反应 (PCR) 技术相比，CRISPR 分子诊断技术缺乏高通量自动化仪器和高灵敏度的标准化检测试剂，限制了其在大规模临床应用。在这项研究中，通过将试剂冻干、无提取技术和一次性耗材系统相结合，开发了一种高通量自动化设备。这种创新方法能够在 25 分钟内快速检测 48 个样本，并对临床样本进行定性分析，表现出高灵敏度和特异性。所得结果表明，设计的系统对非洲猪瘟病毒 (ASFV) 的检测限为 0.5 拷贝/μL。作为一个概念验证，开发了一种单管双靶标核酸检测方法，在 45 分钟内对严重急性呼吸综合征冠状病毒-2 (SARS-CoV-2) 的 和 基因的检测限达到 5 拷贝/μL。该方法具有高度特异性、可靠性和稳定性，为 CRISPR 核酸检测技术的临床应用提供了可行的解决方案。
