Research Progress in Single-base Mutation Detection Methods and Applications

doi:10.13560/j.cnki.biotech.bull.1985.2024-0933

Abstract

Abstract:

Single nucleotide mutation refers to a type of genetic mutation in which a single nucleotide in the genome sequence changes, which has been proven to be an important cause of biological organisms' hereditary traits, susceptibility to diseases, and resistance to drugs. It has significant research significance in many fields, including genetics, disease diagnosis, and biological evolution. With the continuous development of nucleic acid detection technology, single nucleotide mutation detection technology provides critical assistance in assisting plant and animal breeding, detecting disease or microbe-related mutation sites, and guiding the use of therapeutic drugs. This review summarizes several common single nucleotide mutation detection methods, briefly introduces the principles, advantages, and limitations of each method, and lists the application situations of the technology in hereditary traits, disease diagnosis, virus detection, food adulteration, plant and animal breeding, and microbial resistance detection. It focuses on describing the rapid detection strategy of single nucleotide mutation based on the CRISPR/Cas system, and elaborates on the application of the system in different fields based on the precise identification of the target type, and analyzes it in combination with nucleic acid amplification technology. It also discusses the application and development trend of single nucleotide mutation detection technology in the future.

Key words: single-base mutation, rapid detection, multiscenario application, CRISPR-Cas

LIU Hua, SONG Jie, ZENG Hai-juan, WANG Jin-bin, QIAN Yun-fang. Research Progress in Single-base Mutation Detection Methods and Applications[J]. Biotechnology Bulletin, 2025, 41(6): 61-70.

Figures/Tables 1

References 72

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平台名称 Platform name	效应蛋白 Effector protein	扩增方式 Amplification	靶标 Target	结果显示 Readout	检测限 LOD	检测时长 Time	参考文献 References
CRISDA	Cas9	链置换扩增 SDA	乳腺癌相关SNPs Breast cancer-associated SNPs	荧光检测 Fluorescence detection	1 aM	~2 h	［43］
CASLFA	Cas9	聚合酶链式反应 PCR	单增李斯特菌 Listeria monocytogenes	侧向层析试纸条 Lateral flow test strips	100 copies	＜1 h	［44］
	Cas9	链置换扩增+滚环扩增 SDA+RCA	大肠杆菌 O157：H7 E. coli O157：H7	荧光检测 Fluorescence detection	40 CFU/mL	~2 h	［45］
HOLMES	Cas12	聚合酶链式反应 PCR	痛风相关SNP Gout SNP	荧光检测 Fluorescence detection	10 aM	~1 h	［47］
DETECTR	LbCas12a	重组酶聚合酶扩增 RPA	人乳头瘤病毒16/18 HPV16/HPV18	荧光检测 Fluorescence detection	1 aM	≤1 h	［48］
	Cas12a	重组酶聚合酶扩增 RPA	炭疽芽孢杆菌 Bacillus anthracis	肉眼观察 Naked eye	1 copy	~90 min	［49］
	Cas12a	环介导等温扩增 LAMP	肠道沙门氏菌毒力基因 Virulence genes of Salmonella enterica	荧光检测 Fluorescence detection	15 copies/μL	~1 h	［50］
RatioCRISPR	Cas12a	重组酶聚合酶扩增 RPA	线粒体DNA mtDNA	荧光检测 Fluorescence detection	15.7 aM	~25 min	［51］
CASMART	Cas12a	重组酶聚合酶扩增 RPA	肺癌相关基因 EGFR L858R Lung cancer-related genes EGFR L858R	荧光检测 Fluorescence detection	0.3 copies/μL	~1 h	［52］
CDetection	AaCas12b	重组酶聚合酶扩增 RPA	花椰菜花叶病毒/人乳头瘤病毒16/18 CaMV/HPV16/HPV18	荧光检测 Fluorescence detection	1 aM	~1 h	［53］
HOLMESv2	Cas12b	环介导等温扩增 LAMP	癌症相关基因HEK293T SNP HEK293T SNP	荧光检测 Fluorescence detection	10^-8 nM	＜2.5 h	［54］
Cas12c-DETECTOR	Cas12c1	重组酶聚合酶扩增 RPA	新冠病毒/人乳头瘤病毒16/18 CoVID2019 SNP/HPV16/HPV18	荧光检测/侧向层析试纸条 Fluorescence detection/lateral flow test strips	-	~2 h	［55］
Cas14-DETECTR	Cas14a	含硫代磷酸酯引物的聚合酶链式反应 PT-PCR	人类HERC2 SNP Human HERC2 SNP	荧光检测 Fluorescence detection	-	~1 h	［56］
	Cas14a	抑制探针置换扩增BDA	人结直肠癌细胞的BRAF基因的SNP Carcinoma of colon and rectum BRAF SNP	荧光检测 Fluorescence detection	10³copies	＜2 h	［57］
SHERLOCK	Cas13a	重组酶聚合酶扩增 RPA	人体健康/寨卡病毒/登革热病毒SNP Human health/ZIKV/DENV SNP	荧光检测 Fluorescence detection	0.1% DNA	＜1 h	［59-60］
	Cas13a	聚合酶链式反应 PCR	红斑石斑鱼神经坏死病毒 RGNNV	荧光检测 Fluorescence detection	10² fM	＜1 h	［61］
	Cas13a	重组酶聚合酶扩增 RPA	番茄斑萎病毒 TSWV	荧光检测 Fluorescence detection	2.26 × 10² copies/μL	＜1 h	［62］
	RfxCas13d	重组酶聚合酶扩增 RPA	水稻黑条矮缩病毒 RBSDV	荧光检测/侧向层析试纸条 Fluorescence detection / lateral flow test strips	1 aM	30-60 min	［63］

平台名称 Platform name	效应蛋白 Effector protein	扩增方式 Amplification	靶标 Target	结果显示 Readout	检测限 LOD	检测时长 Time	参考文献 References
CRISDA	Cas9	链置换扩增 SDA	乳腺癌相关SNPs Breast cancer-associated SNPs	荧光检测 Fluorescence detection	1 aM	~2 h	［43］
CASLFA	Cas9	聚合酶链式反应 PCR	单增李斯特菌 Listeria monocytogenes	侧向层析试纸条 Lateral flow test strips	100 copies	＜1 h	［44］
	Cas9	链置换扩增+滚环扩增 SDA+RCA	大肠杆菌 O157：H7 E. coli O157：H7	荧光检测 Fluorescence detection	40 CFU/mL	~2 h	［45］
HOLMES	Cas12	聚合酶链式反应 PCR	痛风相关SNP Gout SNP	荧光检测 Fluorescence detection	10 aM	~1 h	［47］
DETECTR	LbCas12a	重组酶聚合酶扩增 RPA	人乳头瘤病毒16/18 HPV16/HPV18	荧光检测 Fluorescence detection	1 aM	≤1 h	［48］
	Cas12a	重组酶聚合酶扩增 RPA	炭疽芽孢杆菌 Bacillus anthracis	肉眼观察 Naked eye	1 copy	~90 min	［49］
	Cas12a	环介导等温扩增 LAMP	肠道沙门氏菌毒力基因 Virulence genes of Salmonella enterica	荧光检测 Fluorescence detection	15 copies/μL	~1 h	［50］
RatioCRISPR	Cas12a	重组酶聚合酶扩增 RPA	线粒体DNA mtDNA	荧光检测 Fluorescence detection	15.7 aM	~25 min	［51］
CASMART	Cas12a	重组酶聚合酶扩增 RPA	肺癌相关基因 EGFR L858R Lung cancer-related genes EGFR L858R	荧光检测 Fluorescence detection	0.3 copies/μL	~1 h	［52］
CDetection	AaCas12b	重组酶聚合酶扩增 RPA	花椰菜花叶病毒/人乳头瘤病毒16/18 CaMV/HPV16/HPV18	荧光检测 Fluorescence detection	1 aM	~1 h	［53］
HOLMESv2	Cas12b	环介导等温扩增 LAMP	癌症相关基因HEK293T SNP HEK293T SNP	荧光检测 Fluorescence detection	10^-8 nM	＜2.5 h	［54］
Cas12c-DETECTOR	Cas12c1	重组酶聚合酶扩增 RPA	新冠病毒/人乳头瘤病毒16/18 CoVID2019 SNP/HPV16/HPV18	荧光检测/侧向层析试纸条 Fluorescence detection/lateral flow test strips	-	~2 h	［55］
Cas14-DETECTR	Cas14a	含硫代磷酸酯引物的聚合酶链式反应 PT-PCR	人类HERC2 SNP Human HERC2 SNP	荧光检测 Fluorescence detection	-	~1 h	［56］
	Cas14a	抑制探针置换扩增BDA	人结直肠癌细胞的BRAF基因的SNP Carcinoma of colon and rectum BRAF SNP	荧光检测 Fluorescence detection	10³copies	＜2 h	［57］
SHERLOCK	Cas13a	重组酶聚合酶扩增 RPA	人体健康/寨卡病毒/登革热病毒SNP Human health/ZIKV/DENV SNP	荧光检测 Fluorescence detection	0.1% DNA	＜1 h	［59-60］
	Cas13a	聚合酶链式反应 PCR	红斑石斑鱼神经坏死病毒 RGNNV	荧光检测 Fluorescence detection	10² fM	＜1 h	［61］
	Cas13a	重组酶聚合酶扩增 RPA	番茄斑萎病毒 TSWV	荧光检测 Fluorescence detection	2.26 × 10² copies/μL	＜1 h	［62］
	RfxCas13d	重组酶聚合酶扩增 RPA	水稻黑条矮缩病毒 RBSDV	荧光检测/侧向层析试纸条 Fluorescence detection / lateral flow test strips	1 aM	30-60 min	［63］

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