CRISPR/Cas核酸检测系统的研究进展和未来挑战

doi:10.3969/j.issn.1673-8640.2024.06.017

摘要/Abstract

摘要：

成簇规律间隔短回文重复序列（CRISPR）/成簇规律间隔短回文重复序列相关蛋白（Cas）系统是一种新兴的分子诊断技术，具有检测速度快、操作简单、特异性高等优势，目前已成为分子诊断领域的研究热点。基于CRISPR/Cas系统构建的核酸检测系统中有一部分已得到临床验证，并获得预期的结果，有望成为一种理想的核酸分子诊断技术，但其在临床大规模应用前尚有一系列问题需要解决。文章对已建立的CRISPR/Cas核酸检测系统的原理、特点和存在的问题进行综述，以期为CRISPR/Cas核酸检测系统的临床应用提供依据。

关键词: 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, 分子诊断技术, 核酸

Abstract:

Clustered regularly interspaced short palindromic repeat（CRISPR）/clustered regularly interspaced short palindromic repeat-associated protein（Cas）nucleic acid diagnosis system，as a new molecular diagnosis technology has advantages of rapid determination，simple operation and high specificity. CRISPR/Cas has been a research hotspot in nucleic acid molecular diagnosis. Some CRISPR/Cas nucleic acid diagnosis systems have been validated by clinical samples，which are expected to be an ideal nucleic acid molecular diagnosis technology. However，there are still a series of problems to be solved before the clinical large-scale commercial application. In this review，the principle，characteristics and problems are summarized，and it is expected to provide a reference for the early clinical application of CRISPR/Cas nucleic acid diagnosis system.

Key words: Clustered regularly interspaced short palindromic repeat, Clustered regularly interspaced short palindromic repeat-associated protein, Molecular diagnosis technology, Nucleic acid

中图分类号:

R446.7

周建, 任雪梅, 王馨, 李卓. CRISPR/Cas核酸检测系统的研究进展和未来挑战[J]. 检验医学, 2024, 39(6): 608-614.

ZHOU Jian, REN Xuemei, WANG Xin, LI Zhuo. Research progress and challenges on CRISPR/Cas nucleic acid diagnosis system[J]. Laboratory Medicine, 2024, 39(6): 608-614.

图/表 4

参考文献 34

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核酸检测系统	Cas蛋白	信号扩增	检测时间	检测下限	检测目的基因	参考文献
NASBACC	SpCas9	LAMP	≤3 h	fmol·L^-1	寨卡病毒	[7]
ctPCR3.0	SpCas9	qPCR	≤2 h	2 ng·L^-1	HPV 16/18型	[10]
Cas12aVDet	EnGenLba、Cas12a	RPA	≤30 min	amol·L^-1	支原体	[17]
CRISPR-ENHANCE	Cas12a	LAMP/ RT-LAMP	60~90 min	fmol·L^-1	SARS-CoV-2	[33]
SHERLOCK+HUDSON	LwCas13a	RPA	<2 h	amol·L^-1	寨卡病毒	[26]
SHERLOCKv2	CcaCas13b、PsmCas13b、LwaCas13a、AsCas12a	RPA	0.5~3 h	zmol·L^-1	寨卡病毒、金黄色葡萄球菌	[28]
CARMEN-Cas13a	LwCas13a	PCR/RPA	-	amol·L^-1	169种与人类相关病毒	[29]

核酸检测系统	Cas蛋白	信号扩增	检测时间	检测下限	检测目的基因	参考文献
NASBACC	SpCas9	LAMP	≤3 h	fmol·L^-1	寨卡病毒	[7]
ctPCR3.0	SpCas9	qPCR	≤2 h	2 ng·L^-1	HPV 16/18型	[10]
Cas12aVDet	EnGenLba、Cas12a	RPA	≤30 min	amol·L^-1	支原体	[17]
CRISPR-ENHANCE	Cas12a	LAMP/ RT-LAMP	60~90 min	fmol·L^-1	SARS-CoV-2	[33]
SHERLOCK+HUDSON	LwCas13a	RPA	<2 h	amol·L^-1	寨卡病毒	[26]
SHERLOCKv2	CcaCas13b、PsmCas13b、LwaCas13a、AsCas12a	RPA	0.5~3 h	zmol·L^-1	寨卡病毒、金黄色葡萄球菌	[28]
CARMEN-Cas13a	LwCas13a	PCR/RPA	-	amol·L^-1	169种与人类相关病毒	[29]

项目	检测下限/（拷贝·μL^-1）	检测时间/min	是否需要特殊设备	反应条件/℃	POCT	特异性
CRISPR/Cas	10	20~180	否	37~42	容易	高
PCR	1	≤240	是	60~95	困难	高

项目	检测下限/（拷贝·μL^-1）	检测时间/min	是否需要特殊设备	反应条件/℃	POCT	特异性
CRISPR/Cas	10	20~180	否	37~42	容易	高
PCR	1	≤240	是	60~95	困难	高