基于CRISPR/Cas系统的生物传感器在动物疫病诊断中的应用

doi:10.11843/j.issn.0366-6964.2024.07.008

摘要/Abstract

摘要：

动物疫病不仅严重危害动物健康，阻碍养殖业良性发展，而且对人类健康构成潜在威胁。开发灵敏、特异、快速的诊断方法对于疫病防控尤为重要。PCR是目前检测病原体核酸的金标准，但是它存在检测周期较长，需要精密仪器和专业技术人员，无法应用于现场即时检测等局限性。成簇规律间隔短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein, CRISPR/Cas)具有敏感性高、特异性强和操作简便等优点，可为动物疫病诊断提供新的方法和契机。CRISPR-Cas9靶向核酸的特异性和CRISPR-Cas12/Cas13“附属切割”活性使其在核酸检测中显示出巨大的应用前景。本文简述了CRISPR/Cas系统的分类，对3种常用的CRISPR/Cas系统的核酸检测策略进行系统阐述，并对其在动物疫病诊断中应用的最新研究进展进行综述，最后讨论了它的优势、面临的挑战及未来发展方向，以期为动物疫病诊断新方法的开发提供参考。

关键词: CRISPR/Cas, 生物传感器, 动物疫病, 检测

Abstract:

Animal diseases not only seriously jeopardize the health of animals and hinder the healthy development of the breeding industry, but also pose a potential threat to human health. Therefore, it is vital to develop sensitive, specific and rapid diagnostic methods for disease prevention and control. The current gold standard for pathogens detection, PCR, has limitations such as long operational cycles, high instrumentation and technician requirements, cannot be applied to point-of-care testing. Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas) have the advantages of high sensitivity, high specificity and easy operation, which can provide a new method and opportunity for the diagnosis of animal diseases. CRISPR-Cas9 specificity in targeting nucleic acids and "collateral cleavage" activity of CRISPR-Cas12/Cas13 show significant promise in nucleic acid detection. In this review, the classification of CRISPR/Cas systems, the strategies adopted by three commonly used CRISPR/Cas systems applied to nucleic acid detection were summarized, as well as the latest research progress of their application in animal disease diagnosis, finally their advantages, challenges, and future development were analyzed, in the hope of providing a reference for the application of CRISPR/Cas biosensing platforms in animal diseases diagnosis.

Key words: CRISPR/Cas, biosensor, animal diseases, detection

中图分类号:

S854.43

陈秀琴, 林甦, 张世忠, 郑敏, 黄梅清. 基于CRISPR/Cas系统的生物传感器在动物疫病诊断中的应用[J]. 畜牧兽医学报, 2024, 55(7): 2859-2876.

Xiuqin CHEN, Su LIN, Shizhong ZHANG, Min ZHENG, Meiqing HUANG. Application of CRISPR/Cas-based Biosensors for Animal Diseases Diagnosis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2859-2876.

图/表 6

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CRISPR/Cas系统在细菌及真菌性疫病诊断中的应用"

靶标 Target	效应蛋白 Effector protein	扩增方法 Amplification	信号输出 Signal output	敏感性 Sensitivity	检测时间 Time	POCT	特点 Characteristic	参考文献 References
耐甲氧西林金黄色葡萄球菌 Methicillin-resistant Staphylococcus aureus	dCas9	无	F	10 CFU·mL^-1	＜30 min	是	采用dCas9/sgRNA复合物作为靶向材料，SYBR Green I作为荧光探针；无需使用细胞裂解物进行基因分离步骤	[68]
产单核细胞李氏杆菌 Listeria monocytogenes	LbCas12a	RPA	E	0.68×10^-18 mol·L^-1	＜2 h	否	-	[65]
副溶血性弧菌 Vibrio parahaemolyticus	LbCas12a	LAMP	F	30 copies·反应^-1	50 min	是	离心式芯片耦合CRISPR/Cas12a系统的集成核酸检测方法	[69]
副溶血性弧菌 Vibrio parahaemolyticus	LbCas12a	RPA	F	12.3 CFU·mL^-1	-	否	一个RPA扩增产物可以激活多CRISPR/Cas12a单元；克服了PAM限制的局限性	[70]
沙门菌 Salmonella	LbCas12a	无	C	1 CFU·mL^-1	＜3 h	是	利用无标记G-四链体作为报告系统；结果既可以用肉眼识别，也可以用智能手机辅助定量测量	[71]
鼠伤寒沙门菌 Salmonella Typhimurium	LbCas12a	LAMP	F	1 CFU·mL^-1	~2 h	否	首次将比率型荧光信号读数与CRISPR/Cas结合起来; 使用无标记DNA模板的银纳米簇将目标核酸信号转换为双色荧光	[72]
鼠伤寒沙氏菌 Salmonella Typhimurium	LbCas12a	末端脱氧核苷酸转移酶(Tdt)介导的信号放大	F	53 CFU·mL^-1	-	否	率先将尿嘧啶-DNA糖基化酶(UDG)消化技术与V型PCR(VPCR)相结合；首次将Tdt介导的信号放大技术用于预制备的多聚磁探针；构建的UDG-VPCR驱动的Cas12a-MRS(磁弛豫开关)具有四重特异性；解决了与核酸扩增相结合的Cas12a系统相关的气溶胶污染问题	[73]
7种食源性致病菌 Seven food-borne pathogens	LbCas12a	RPA	F	＜500 CFU·mL^-1	~1 h	是	开发了一种基于单管CRISPR/Cas12a-RPA的手指驱动微流体生物传感器；同时检测7种致病菌；解决了气溶胶污染问题	[74]
7种食源性致病菌 Seven food-borne pathogens	LbCas13a	RAA	液滴微流控	10 copies·μL^-1	~1 h	否	开发了一种基于单管CRISPR/Cas13a-RAA的液滴微流控平台，目标细菌鉴定通过液滴的颜色确定，通过泊松分布计算阳性液滴的百分比来计算目标细菌浓度。解决了气溶胶污染问题，且定量更准确	[75]
金黄色葡萄球菌 Staphylococcus aureus	LbCas12a	SDA	ECL	0.437×10^-18 mol·L^-1	-	否	卟啉Zr金属有机骨架作为共反应促进剂加速了电子转移并提高了ECL发光效率；设计有缺陷的T型连接结构用于SDA，消除了对目标DNA长度的考虑，具有更高的特异性和稳定性	[76]
炭疽杆菌 Bacillus anthracis	LbCas12a	RPA	F	2 copies·反应^-1	40 min	是	-	[77]
布鲁氏菌 Brucella spp.	LbCas12a	RPA	F/E	2 copies·反应^-1	-	否	-	[78]
幽门螺杆菌 Helicobacter pylori	LbCas12a	MIRA	F/LFA	1 copies·反应^-1 (F)；4 copies·反应^-1	45 min	是	-	[79]
犬小孢子菌和须癣毛癣菌 Microsporum canis, Trichophyton mentagrophytes	LbCas12a	RPA	F	100%	~30 min	是	-	[64]
念珠菌、曲霉菌和隐球菌 Candida, Aspergillus, Cryptococcus	LbCas12a	RT-RPA	纸基微流控	＜10 CFU·mL^-1	-	是	通过DNA智能水凝胶体系和葡萄糖氧化酶-辣根过氧化物酶级联反应进行信号的放大与转导，并最终利用纸基微流控芯片进行肉眼读取结果	[80]

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亚型Subtypes	Ⅱ型Type Ⅱ	Ⅴ型Type Ⅴ		Ⅵ型Type Ⅵ
效应蛋白 Effector protein	Cas9	Cas12a	Cas14a/b	Cas13a/b
核酸酶结构域 Nuclease domains	RuvC、HNH	RuvC	RuvC	HEPN^a
向导RNA Guide RNA	tracrRNA^b、crRNA^c	crRNA	tracrRNA、crRNA	crRNA
间隔区长度/nt Spacer length	18~24	18~25	20~40	22~30
PAM/PFS	3′ GC-rich PAM^d	5′ AT-rich PAM	-	3′ PFS^e: non-G
结合的靶标 Target binding	dsDNA	DNA (ds/ss^f)	DNA (ds/ss)	ssRNA
反式切割底物 Trans-cleavage substrates	-	ssDNA	ssDNA	ssRNA

检测方法 Detection method	敏感性 Sensitivity	特异性 Specificity	检测速度 Speed	POCT	可操作性 Operability	检测病原体的广度 Breadth of pathogens	成本 Cost
PCR	高	较高	h	否	中等	广	中等
等温扩增^a Isothermal amplification	中等	中等	min~h	是	容易	广	较高
基因测序 Gene sequencing	中等	高	数小时至数天	否	最难	无限	高
CRISPR/Cas	可以很高	高	min~h	是	较容易	广	可以很低

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