Establishment of a CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Assay for the Rapid, Sensitive, and Specific Detection of Brucella ovis.

IF 3.8 2区 医学 Q2 CHEMISTRY, MEDICINAL
Yue Zhang, Xinggui Yang, Yue Wang, Fengming Chen, Ying Liu, Hai Jiang, Yi Wang, Yong Hu, Shijun Li
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引用次数: 0

Abstract

Brucella ovis (B. ovis), a major pathogenic species within the Brucella genus, causes ovine epididymitis. Although the isolation and identification of B. ovis remain the gold standard for diagnosis, these methods are unsuitable for early detection. The traditional polymerase chain reaction (PCR) offers faster detection but requires specialized equipment such as PCR thermal cyclers and gel electrophoresis imagers, limiting its use in basic laboratories. Thus, developing rapid, sensitive, and specific diagnostic strategies is vital for preventing and controlling the spread of ovine brucellosis. In this study, we developed a diagnostic assay combining clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b with multiple cross displacement amplification (MCDA)─termed CRISPR/Cas12b-MCDA─for rapid, sensitive, and specific identification of B. ovis. In the CRISPR/Cas12b-MCDA system, MCDA amplicons containing protospacer adjacent motif (PAM) sites are recognized by the Cas12b/gRNA complex, which binds the target region and triggers trans-cleavage of a single-stranded DNA (ssDNA) reporter. The CRISPR/Cas12b-MCDA assay demonstrated a detection limit of 10 fg/μL for synthetic genomic DNA and exhibited 100% specificity for B. ovis, with no cross-reactivity against other Brucella or non-Brucella species. The preamplification for template extraction takes 20 min, then 5 min for uracil DNA glycosylase (UDG) digestion, and 45 min for MCDA amplification. The total detection time was 75 min using real-time fluorescence analysis and 90 min with a lateral flow biosensor (LFB). Additionally, the results were validated using UV visualization to confirm the CRISPR/Cas12b-MCDA results. Notably, both LFB and UV analyses are instrument-free, enhancing their accessibility. In conclusion, the CRISPR/Cas12b-MCDA assay is a simple, rapid, sensitive, specific, and reliable method for detecting B. ovis.

基于CRISPR/ cas12b的多重交叉位移扩增快速、灵敏、特异检测布鲁氏菌方法的建立
羊布鲁氏菌(B. ovis)是布鲁氏菌属中的一种主要致病菌,可引起绵羊附睾炎。虽然分离和鉴定卵巢双歧杆菌仍然是诊断的金标准,但这些方法不适合早期发现。传统的聚合酶链式反应(PCR)提供了更快的检测,但需要专门的设备,如PCR热循环仪和凝胶电泳成像仪,限制了它在基础实验室的使用。因此,制定快速、敏感和具体的诊断策略对于预防和控制牛布鲁氏菌病的传播至关重要。在这项研究中,我们开发了一种将聚集规律间隔短回文重复序列(CRISPR)/Cas12b与多重交叉位移扩增(MCDA)(称为CRISPR/Cas12b-MCDA)相结合的诊断方法,用于快速、敏感和特异性地鉴定B. ovis。在CRISPR/Cas12b-MCDA系统中,含有原间隔器邻近基元(PAM)位点的MCDA扩增子被Cas12b/gRNA复合体识别,结合靶区并触发单链DNA (ssDNA)报告基因的反式切割。CRISPR/Cas12b-MCDA分析显示,合成基因组DNA的检出限为10 fg/μL,对B. ovis具有100%的特异性,与其他布鲁氏菌或非布鲁氏菌没有交叉反应性。模板提取预扩增时间为20 min, UDG酶切时间为5 min, MCDA扩增时间为45 min。实时荧光法检测总时间为75分钟,横向流动生物传感器(LFB)检测总时间为90分钟。此外,使用UV可视化验证结果以确认CRISPR/Cas12b-MCDA结果。值得注意的是,LFB和UV分析都是不需要仪器的,这提高了它们的可访问性。综上所述,CRISPR/Cas12b-MCDA检测方法简便、快速、灵敏、特异、可靠。
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来源期刊
ACS Infectious Diseases
ACS Infectious Diseases CHEMISTRY, MEDICINALINFECTIOUS DISEASES&nb-INFECTIOUS DISEASES
CiteScore
9.70
自引率
3.80%
发文量
213
期刊介绍: ACS Infectious Diseases will be the first journal to highlight chemistry and its role in this multidisciplinary and collaborative research area. The journal will cover a diverse array of topics including, but not limited to: * Discovery and development of new antimicrobial agents — identified through target- or phenotypic-based approaches as well as compounds that induce synergy with antimicrobials. * Characterization and validation of drug target or pathways — use of single target and genome-wide knockdown and knockouts, biochemical studies, structural biology, new technologies to facilitate characterization and prioritization of potential drug targets. * Mechanism of drug resistance — fundamental research that advances our understanding of resistance; strategies to prevent resistance. * Mechanisms of action — use of genetic, metabolomic, and activity- and affinity-based protein profiling to elucidate the mechanism of action of clinical and experimental antimicrobial agents. * Host-pathogen interactions — tools for studying host-pathogen interactions, cellular biochemistry of hosts and pathogens, and molecular interactions of pathogens with host microbiota. * Small molecule vaccine adjuvants for infectious disease. * Viral and bacterial biochemistry and molecular biology.
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