单碱基突变检测方法及应用的研究进展

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

摘要/Abstract

摘要：

单碱基突变是指在基因组序列中由于单个核苷酸发生改变的一种基因突变类型，已被证明是造成生物体遗传性状、疾病易感性和耐药性的重要原因之一，在遗传学、疾病诊断及生物进化等众多领域具有重要研究意义。随着核酸检测技术的不断发展，单碱基突变检测技术为辅助动植物育种、检测疾病或微生物相关突变位点及指导治疗药物使用提供关键助力。本文综述了几种常见的单碱基突变检测方法，简要介绍了各种方法的原理、优势及局限性，列举了该技术在遗传性状、疾病诊断、病毒检测、食品掺假、动植物育种以及微生物耐药性检测等方面的应用情况。重点描述了基于CRISPR/Cas系统的单碱基突变快速检测策略，依据精准识别靶标类型不同，对该系统在不同领域的应用进行阐述，同时结合无核酸扩增技术进行分析，并对未来单碱基突变检测技术的应用及发展趋势进行了探讨，以期为开发快速且经济的单碱基突变检测技术提供思路。

关键词: 单碱基突变, 快速检测, 多场景应用, CRISPR-Cas

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

刘华, 宋洁, 曾海娟, 王金斌, 钱韻芳. 单碱基突变检测方法及应用的研究进展[J]. 生物技术通报, 2025, 41(6): 61-70.

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.

图/表 1

参考文献 72

1	范广轩, 王洪亮, 邢秀梅. SNP标记的研究进展及其应用 [J/OL]. 特产研究, 2023: 1-9. (2023-11-29). .
	Fan GX, Wang HL, Xing XM. Advances in SNP marker research and its applications [J/OL]. China Ind Econ, 2023: 1-9. (2023-11-29). .
2	Sachidanandam R, Weissman D, Schmidt SC, et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms [J]. Nature, 2001, 409(6822): 928-933.
3	Jorde LB. Linkage disequilibrium and the search for complex disease genes [J]. Genome Res, 2000, 10(10): 1435-1444.
4	Giampaoli S, Chillemi G, Valeriani F, et al. The SNPs in the human genetic blueprint era [J]. New Biotechnol, 2013, 30(5): 475-484.
5	苏睿, 林峻, 陈鲤群, 等. 高通量自动化SNP检测技术研究进展 [J]. 中国细胞生物学学报, 2019, 41(7): 1412-1422.
	Su R, Lin J, Chen LQ, et al. Research progress on high-throughput automated SNP detection technology [J]. Chin J Cell Biol, 2019, 41(7): 1412-1422.
6	Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors [J]. Proc Natl Acad Sci USA, 1977, 74(12): 5463-5467.
7	张微, 冯琳琳, 平昭. 单核苷酸多态性检测技术研究进展 [J]. 生物技术通讯, 2016, 27(6): 879-883.
	Zhang W, Feng LL, Ping Z. Progress in technologies for single nucleotide polymorphism detection [J]. Lett Biotechnol, 2016, 27(6): 879-883.
8	Bloemen M, Rector A, Swinnen J, et al. Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing [J]. J Virol Methods, 2022, 304: 114512.
9	Liu GL, Xin SY, Geng S, et al. Identification of a novel fusion gene NLRC3-NLRP12 in miiuy croaker (Miichthys miiuy) [J]. Fish Shellfish Immunol, 2023, 136: 108697.
10	Royo JL, Galán JJ. Pyrosequencing for SNP genotyping [M]//Single Nucleotide Polymorphisms. Totowa, NJ: Humana Press, 2009: 123-133.
11	Ortola-Vidal A, Schnerr H, Rojmyr M, et al. Quantitative identification of plant Genera in food products using PCR and Pyrosequencing^® technology [J]. Food Contr, 2007, 18(8): 921-927.
12	Ramünke S, Melville L, Rinaldi L, et al. Benzimidazole resistance survey for Haemonchus, Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus [J]. Int J Parasitol Drugs Drug Resist, 2016, 6(3): 230-240.
13	Park JW, Park IH, Kim JM, et al. Rapid detection of FMO3 single nucleotide polymorphisms using a pyrosequencing method [J]. Mol Med Rep, 2022, 25(2): 48.
14	Erali M, Wittwer CT. High resolution melting analysis for gene scanning [J]. Methods, 2010, 50(4): 250-261.
15	Vishnuraj MR, Renuka J, Aravind Kumar N, et al. Differential detection of sheep and goat meat using duplex real-time PCR and high-resolution melt analysis [J]. Food Chem Adv, 2023, 2: 100309.
16	Forghani F, Wei S, Oh DH. A rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in food [J]. J Food Prot, 2016, 79(5): 810-815.
17	Liu YJ, Singh P, Mustapha A. High-resolution melt curve PCR assay for specific detection of E. coli O157: H7 in beef [J]. Food Contr, 2018, 86: 275-282.
18	Gibriel AA, Adel O. Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications [J]. Mutat Res, 2017, 773: 66-90.
19	Ruiz C, Huang J, Giardina S F, et al. Single-molecule detection of cancer mutations using a novel PCR-LDR-qPCR assay [J]. Hum Mutat, 2020, 41(5): 1051-1068.].
20	Wang YZ, Xiao JH, Liu LG, et al. Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction [J]. Braz J Infect Dis, 2011, 15(6): 560-566.
21	Li HF, Shu JT, Du YF, et al. Analysis of the genetic effects of prolactin gene polymorphisms on chicken egg production [J]. Mol Biol Rep, 2013, 40(1): 289-294.
22	Yang ZX, Lo YT, Quan Z, et al. Application of a modified Tetra-primer ARMS-PCR assay for rapid Panax species identity authentication in ginseng products [J]. Sci Rep, 2023, 13(1): 14396.
23	Yang H, Yang S, Xia XH, et al. Sensitive detection of a single-nucleotide polymorphism in foodborne pathogens using CRISPR/Cas12a-signaling ARMS-PCR [J]. J Agric Food Chem, 2022, 70(27): 8451-8457.
24	Xie YY, Ping Y, Yu P, et al. The rs9402373 polymorphism of CTGF gene may not be related to inflammatory bowel disease susceptibility in Chinese population based on ARMS-PCR genotyping [J]. Heliyon, 2023, 9(6): e17003.
25	Ho UH, Pak SH, Kim K, et al. Efficient screening of SNP in canine OR52N9 and OR9S25 as assistant marker of olfactory ability [J]. J Vet Behav, 2023, 60: 51-55.
26	Honardoost MA, Tabatabaeian H, Akbari M, et al. Investigation of sensitivity, specificity and accuracy of Tetra primer arms pcr method in comparison with conventional arms pcr, based on sequencing technique outcomes in ivs-ii-i genotyping of beta thalassemia patients [J]. Gene, 2014, 549(1): 1-6.
27	Wang ZN, Li MJ, Lan XY, et al. Tetra-primer ARMS-PCR identifies the novel genetic variations of bovine HNF-4α gene associating with growth traits [J]. Gene, 2014, 546(2): 206-213.
28	Xu XY, Hu XG, Ma GD, et al. Detecting fa leptin receptor mutation in Zucker rats with Tetra-primer amplification-refractory mutation system (ARMS)-PCR [J]. Heliyon, 2023, 9(9): e20159.
29	Ryu WS. Diagnosis and methods [M]//Molecular Virology of Human Pathogenic Viruses. Amsterdam: Elsevier, 2017: 47-62.
30	Boccacci P, Chitarra W, Schneider A, et al. Single-nucleotide polymorphism (SNP) genotyping assays for the varietal authentication of 'Nebbiolo' musts and wines [J]. Food Chem, 2020, 312: 126100.
31	Jing QY, Liu SJ, Song Y, et al. TaqMan real-time quantitative PCR for the detection of beef tallow to assess the authenticity of edible oils [J]. Food Contr, 2024, 156: 110139.
32	Bundidamorn D, Supawasit W, Trevanich S. Taqman^® probe based multiplex RT-PCR for simultaneous detection of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli in foods [J]. LWT, 2021, 147: 111696.
33	Hirotsu Y, Maejima M, Shibusawa M, et al. Classification of Omicron BA.1, BA.1.1, and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data [J]. Int J Infect Dis, 2022, 122: 486-491.
34	Fondevila M, Børsting C, Phillips C, et al. Forensic SNP genotyping with SNaPshot: technical considerations for the development and optimization of multiplexed SNP assays [J]. Forensic Sci Rev, 2017, 29(1): 57-76.
35	Li L, Li CJ, Zhang YJ, et al. Simultaneous detection of CYP3A5 and MDR1 polymorphisms based on the SNaPshot assay [J]. Clin Biochem, 2011, 44(5-6): 418-422.
36	Paneto GG, Köhnemann S, Martins JA, et al. A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups [J]. Mitochondrion, 2011, 11(2): 296-302.
37	Zhang B, Zhao N, Peng KK, et al. A combination of genome-wide association study screening and SNaPshot for detecting sex-related SNPs and genes in Cynoglossus semilaevis [J]. Comp Biochem Physiol Part D Genomics Proteomics, 2020, 35: 100711.
38	Yoo E, Haile M, Ko HC, et al. Development of SNP markers for Cucurbita species discrimination [J]. Sci Hortic, 2023, 318: 112089.
39	Huang CH, Chang MT, Huang MC, et al. Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group [J]. Mol Cell Probes, 2011, 25(4): 153-157.
40	Cui YB, Xu JM, Cheng MX, et al. Review of CRISPR/Cas9 sgRNA design tools [J]. Interdiscip Sci, 2018, 10(2): 455-465.
41	Hillary VE, Ceasar SA. A review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering [J]. Mol Biotechnol, 2023, 65(3): 311-325.
42	Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering [J]. Science, 2018, 361(6405): 866-869.
43	Zhou WH, Hu L, Ying LM, et al. A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection [J]. Nat Commun, 2018, 9(1): 5012.
44	Wang XS, Xiong EH, Tian T, et al. Clustered regularly interspaced short palindromic repeats/Cas9-mediated lateral flow nucleic acid assay [J]. ACS Nano, 2020, 14(2): 2497-2508.
45	Sun X, Wang Y, Zhang L, et al. CRISPR-Cas9 triggered two-step isothermal amplification method for E. coli O157: H7 detection based on a metal-organic framework platform [J]. Anal Chem, 2020, 92(4): 3032-3041.
46	Yan WX, Hunnewell P, Alfonse LE, et al. Functionally diverse type V CRISPR-cas systems [J]. Science, 2019, 363(6422): 88-91.
47	Li SY, Cheng QX, Wang JM, et al. CRISPR-Cas12a-assisted nucleic acid detection [J]. Cell Discov, 2018, 4: 20.
48	Chen JS, Ma EB, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity [J]. Science, 2018, 360(6387): 436-439.
49	Wang DS, Chen G, Lyu YF, et al. A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis [J]. Emerg Microbes Infect, 2022, 11(1): 428-437.
50	Zhu YY, Liu JL, Liu SN, et al. CRISPR/Cas12a-assisted visible fluorescence for pseudo dual nucleic acid detection based on an integrated chip [J]. Anal Chim Acta, 2023, 1280: 341860.
51	Wu XL, Zhao Y, Guo CH, et al. RatioCRISPR: a ratiometric biochip based on CRISPR/Cas12a for automated and multiplexed detection of heteroplasmic SNPs in mitochondrial DNA [J]. Biosens Bioelectron, 2023, 241: 115676.
52	Zhang CQ, Cai ZY, Zhou ZH, et al. CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles [J]. Biosens Bioelectron, 2023, 222: 114956.
53	Teng F, Guo L, Cui TT, et al. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity [J]. Genome Biol, 2019, 20(1): 132.
54	Li LX, Li SY, Wu N, et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation [J]. ACS Synth Biol, 2019, 8(10): 2228-2237.
55	Wang ZP, Zhong CH. Cas12c-DETECTOR: a specific and sensitive Cas12c-based DNA detection platform [J]. Int J Biol Macromol, 2021, 193(Pt A): 441-449.
56	Harrington LB, Burstein D, Chen JS, et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes [J]. Science, 2018, 362(6416): 839-842.
57	He YW, Shao SJ, Chen JH. High-fidelity identification of single nucleotide polymorphism by type V CRISPR systems [J]. ACS Sens, 2023, 8(12): 4478-4483.
58	Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector [J]. Science, 2016, 353(6299): aaf5573.
59	Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2 [J]. Science, 2017, 356(6336): 438-442.
60	Kellner MJ, Koob JG, Gootenberg JS, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases [J]. Nat Protoc, 2019, 14(10): 2986-3012.
61	Huang FQ, Shan JH, Liang KS, et al. A new method to detect red spotted grouper neuro necrosis virus (RGNNV) based on CRISPR/Cas13a [J]. Aquaculture, 2022, 555: 738217.
62	Zhang WH, Jiao YB, Ding CY, et al. Rapid detection of tomato spotted wilt virus with Cas13a in tomato and Frankliniella occidentalis [J]. Front Microbiol, 2021, 12: 745173.
63	Li LN, Duan CX, Weng JF, et al. A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13 [J]. Sci China Life Sci, 2022, 65(7): 1456-1465.
64	Kang TJ, Lu JM, Yu T, et al. Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example [J]. Biosens Bioelectron, 2022, 206: 114109.
65	Guk K, Keem JO, Hwang SG, et al. A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex [J]. Biosens Bioelectron, 2017, 95: 67-71.
66	Balderston S, Taulbee JJ, Celaya E, et al. Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor [J]. Nat Biomed Eng, 2021, 5(7): 713-725.
67	Zhu YD, Lin YN, Gong B, et al. Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping [J]. Biosens Bioelectron, 2024, 255: 116255.
68	Hu YB, Liao YW, Pan ST, et al. A Triple-Mismatch Differentiating assay exploiting activation and trans cleavage of CRISPR-Cas12a for mutation detection with ultra specificity and sensitivity [J]. Biosens Bioelectron, 2025, 267: 116826.
69	Jiang T, Guo HZ, Liu YD, et al. A comprehensive genetic variant reference for the Chinese population [J]. Sci Bull, 2024, 69(24): 3820-3825.
70	Newby GA, Yen JS, Woodard KJ, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice [J]. Nature, 2021, 595(7866): 295-302.
71	Wang MY, Liu XJ, Yang JT, et al. CRISPR/Cas12a-based biosensing platform for the on-site detection of single-base mutants in gene-edited rice [J]. Front Plant Sci, 2022, 13: 944295.
72	Köbölkuti ZA, Cseke K, Benke A, et al. Allelic variation in candidate genes associated with wood properties of cultivated poplars (Populus) [J]. Biol Futur, 2019, 70(4): 286-294.

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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