Xu Hongpan, Peng Lijun, Wu Jie, Khan Adeel, Sun Yifan, Shen Han, Li Zhiyang
Nanjing Drum Tower Hospital Clinical College of Jiangsu University, Nanjing, China.
Clinical Laboratory Center, Affiliated Hangzhou Chest Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Front Bioeng Biotechnol. 2023 Jan 5;10:1094028. doi: 10.3389/fbioe.2022.1094028. eCollection 2022.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo (3.3.1.13,7) decan]-4-yl}-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30 min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7 fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity.
成簇规律间隔短回文重复序列（CRISPR）和成簇规律间隔短回文重复序列相关蛋白（CRISPR-Cas）在核酸分子诊断领域有着广阔的前景。然而，基于成簇规律间隔短回文重复序列的荧光检测技术主要受到具有共轭双键的蛋白质和自发荧光的阻碍，导致荧光背景高、灵敏度低以及反应体系不兼容，不利于临床自动化检测。化学发光（CL）检测技术因其高灵敏度、低背景和快速响应而得到广泛应用。因此，我们开发了一种基于成簇规律间隔短回文重复序列-成簇规律间隔短回文重复序列相关蛋白13a、结合磁珠（MBs）和化学发光（CL）的快速、超灵敏且经济的检测系统（Cas13a-MB-CL），用于检测急性呼吸道传染病甲型H7N9流感病毒。羧基功能化磁珠（MBs-COOH）与胺化RNA探针共价偶联，而RNA探针的另一端用生物素修饰。碱性磷酸酶标记的链霉亲和素（SA-ALP）与生物素结合形成磁珠复合物。在靶RNA存在的情况下，激活Cas13a的旁切活性以降解磁珠上的RNA探针，并从复合物中释放碱性磷酸酶。然后对复合物进行磁分离，随后加入ALP底物2-氯-5-{4-甲氧基螺[1,2-二氧杂环丁烷-3,2'-（5'-氯）三环（3.3.1.13,7）癸烷]-4-基}-1-苯基磷酸二钠（CDP-star），以产生化学发光信号。通过测量化学发光强度来定量相关蛋白13a 的活性和靶RNA的存在。该方法在25℃下30分钟内完成了对H7N9的检测。当与逆转录-重组酶辅助扩增（RT-RAA）结合时，检测下限低至19.7 fM（3S/N）。我们提出的磁珠-相关蛋白13a-化学发光方法进一步用于检测H7N9临床样本，显示出优异 Sensitivity and specificity.（此处英文原文有误，结合前文推测可能是“sensitivity and specificity”，中文为“灵敏度和特异性”）
