可视化分析在病原体核酸现场快速检测中的研究进展

doi:10.16288/j.yczz.22-323

遗传 ›› 2023, Vol. 45 ›› Issue (4): 306-323.doi: 10.16288/j.yczz.22-323

可视化分析在病原体核酸现场快速检测中的研究进展

刘丹妮^1,²(), 武海萍^2,³, 周国华^1,^2,^3,⁴()

1.中国药科大学生命科学与技术学院，南京 210009
2.东部战区总医院临床药学科，南京，210002
3.南方医科大学药学院，广州 510515
4.南京医科大学药学院，南京 211166

收稿日期:2022-12-28 修回日期:2023-02-05 出版日期:2023-04-20 发布日期:2023-02-16
通讯作者: 周国华 E-mail:1370503352@qq.com;ghzhou@nju.edu.cn
作者简介:刘丹妮，在读硕士研究生，专业方向：分子诊断。E-mail: 1370503352@qq.com
基金资助:
中央高校基本科研业务费专项资金资助(2022300326);国家自然科学基金项目(61871403)

Research progress of visual detection in rapid on-site detection of pathogen nucleic acid

Danni Liu^1,²(), Haiping Wu^2,³, Guohua Zhou^1,^2,^3,⁴()

1. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
2. Department of Clinical Pharmacy, General Hospital of Eastern Military Command, Nanjing 210002, China
3. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
4. School of Pharmacy, Nanjing Medical University, Nanjing 211166, China

Received:2022-12-28 Revised:2023-02-05 Online:2023-04-20 Published:2023-02-16
Contact: Zhou Guohua E-mail:1370503352@qq.com;ghzhou@nju.edu.cn
Supported by:
Fundamental Research Funds for the Central Universities(2022300326);National Natural Science Foundation of China(61871403)

摘要/Abstract

摘要：

核酸检测因灵敏度高、特异性强而被广泛应用于病原体筛查与检测。随着检测需求的增加和扩增技术的发展，核酸检测方法逐渐向简单、快速、低成本方向发展。作为核酸检测“金标准”的实时荧光定量PCR法依赖昂贵的荧光读取设备和专业的操作人员，并不适用于病原体的现场快速检测。可视化检测方法无需依赖激发光源或复杂的设备，将可视化检测方法与快速、高效的扩增技术结合，能够以更直观、便携的方式呈现检测结果，具有即时检测(point-of-care testing，POCT)的潜力。本文对与扩增技术、CRISPR/Cas技术结合的可视化分析在病原体核酸快速检测中的应用及优缺点展开综述，以期为基于病原体核酸的POCT策略提供参考。

关键词: 可视化, 病原体检测, 等温扩增, 即时检测, CRISPR/Cas系统

Abstract:

Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.

Key words: visual detection, pathogen detection, isothermal amplification, point-of-care testing, CRISPR/Cas system

刘丹妮, 武海萍, 周国华. 可视化分析在病原体核酸现场快速检测中的研究进展[J]. 遗传, 2023, 45(4): 306-323.

Danni Liu, Haiping Wu, Guohua Zhou. Research progress of visual detection in rapid on-site detection of pathogen nucleic acid[J]. Hereditas(Beijing), 2023, 45(4): 306-323.

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检测指示剂	指示剂类型	检测结果	检测原理
钙黄绿素	金属指示剂	橙色-绿色	钙黄绿素与Mn²⁺结合时反应体系为浅橙色，随着LAMP扩增的进行，扩增副产物PPi会与钙黄绿素竞争性结合Mn²⁺，使钙黄绿素游离，在紫外照射下会发出强烈荧光，自然光下呈现为绿色。
HNB	金属指示剂	紫色-天蓝色	HNB与Mg²⁺结合时反应体系为紫色，随着LAMP扩增的进行，副产物PPi会Mg²⁺结合形成稳定的络合物，导致HNB游离，反应体系由紫色转变为蓝色。
酚红	酸碱指示剂	红色-黄色	酚红变色范围：6.8~8.2，体系小于6.8时呈现为黄色，大于8.2时为红色。LAMP扩增时，氢离子不断积累，反应体系由碱性变为酸性，酚红由红色转为黄色。
中性红	酸碱指示剂	黄色-红色	中性红变色范围：6.4~8.0，体系小于6.4时呈现为红色，大于8.0时为黄色。LAMP扩增时，氢离子不断积累，使反应体系由碱性变为酸性，中性红由黄色转为红色。

可视化检测方法	检测原理	检测病原体	LOD	参考文献
酶促底物显色	在H₂O₂作用下，HRP催化ABTS^2-显色	SARS-CoV-2	0.28 pmol/L	[52]
酶促底物显色	G-四链体/血红素具有类过氧化酶活性，可催化TMB显色	副溶血弧菌	100 CFU/mL	[54]
纳米颗粒显色	利用可以与靶标序列互补的寡核苷酸修饰的AuNPs作为比色报告物，对扩增产物进行特异性识别，促使AuNPs聚集	SARS-CoV-2 N基因	10拷贝/反应	[58]
	通过核酸侵入反应识别多重PCR扩增产物，通过AuNPs杂交反应指示检测结果	HPV分型检测	0.5拷贝/μL	[60]
	通过核酸侵入反应识别多重RT-LAMP扩增产物，通过AuNPs杂交反应指示检测结果	副流感A/H1N1、副流感A/H3、副流感B	10拷贝/反应、 10拷贝/反应、 100拷贝/反应	[61]
横向流动分析	LAMP-LFA方法通过双引物标记、单引物标记、无标签标记的策略构建双标记扩增产物，并将其加载到横向流动试纸条上，通过抗原抗体杂交使信号标签在T线上不断积累	SARS-CoV-2、肺炎链球菌	2拷贝/μL 25 fg/反应	[65,66]
	RPA-Nfo-LFA 通过引入生物素标记RPA引物及Nfo探针构建双标记扩增产物，通过抗原抗体杂交实现信号标签在T线上的累积	肠道病毒、 SARS-CoV-2 寨卡病毒	5拷贝/反应 7.59拷贝/μL 10拷贝/μL	[69,71,72]
	mRT-LAMP +三线横向流动试纸条通过对mRT-LAMP的扩增引物进行不同标记，实现扩增产物的双标记且产物间的标记不同，再加载到三线横向流动试纸条上	SARS-CoV-2的ORF1ab和N基因	12拷贝/反应	[74]

可视化检测方法	CRISPR/ Cas系统	扩增方法	检测原理	检测病原体	检测时长	LOD	参考文献
DNAzyme	Cas9	RT-LAMP	Cas9切割DNAzyme结构，关闭比色信号	SARS-CoV-2 ORF1ab、N、 S基因	50 min	10、9、13拷贝/反应	[79]
DNAzyme	Cas12a	LAMP	被激活的Cas12a切割DNAzyme结构，使显色底物无法显色	副溶血弧菌	150 min	9.8 CFU/反应	[80]
AuNPs	Cas12a	RT-RPA	被激活的Cas12a切割AuNPs上起稳定作用的ssDNA，使AuNPs聚集	SARS-CoV-2	60 min	单拷贝/反应	[81]
	Cas13a	RT-RPA/ T7转录	被激活的Cas12a切割AuNPs上起稳定作用的ssRNA，AuNPs聚集	SARS-CoV-2	120 min	3 fmol/L	[82]
	Cas12a	RT-PCR	被激活的Cas12a切割AuNPs之间的连接子，AuNPs游离，体系呈红色	SARS-CoV-2	90 min	1拷贝/μL	[83]
	Cas12a	RT-RPA	被激活的Cas12a切割AuNPs与磁珠的连接子，AuNPs游离，体系呈红色	SARS-CoV-2	90 min	50拷贝/反应	[84]
横向流动分析法	Cas13a	RT-RPA/ T7转录	被激活的Cas13a切割ssRNA报告分子，再进行LFA	SARS-CoV-2、猪流行性腹泻病毒	50 min、 45 min	100拷贝/μL、10拷贝/反应	[85,86]
横向流动分析法	Cas12a	RT-LAMP	被激活的Cas12a切割ssDNA报告分子，再进行LFA	SARS-CoV-2	60 min	1拷贝/μL	[87]
多重横向流动分析	Cas9	mRT-RPA	通过在Cas9系统的gRNA序列中保留AuNPs-DNA探针识别区域，实现AuNPs-DNA探针T线上的不断聚集	SARS-CoV-2 E 和ORF1ab基因	60 min	100拷贝/反应	[88]
多重横向流动分析	Cas12a+ Cas13a	RT-RPA+ RT-RPA/ T7转录	利用Cas12a和Cas13a的不同的切割偏好切割相应的报告分子后加载到三线横向流动试纸条	SARS-CoV-2	60 min	10拷贝/反应	[89]

可视化检测方法	检测原理	检测结果	优点	缺点
非特异性的可视化检测方法
比浊法	LAMP副产物焦磷酸根离子与体系中的Mg²⁺结合，生成不溶于水的白色沉淀。	阴：体系澄清阳：体系浑浊/离心后有白色沉淀	成本低廉、操作简单、可实时监测	白色沉淀不易观察、灵敏度低；非特异性检测
指示剂显色法	金属指示剂：因LAMP扩增导致游离的Mg²⁺浓度下降使指示剂呈现不同颜色。	以HNB为例：阴：紫阳：蓝	操作简单、成本低廉	对比效应不明显，易出现结果误读；非特异性检测
指示剂显色法	酸碱指示剂：因LAMP扩增导致的H⁺浓度上升而指示剂结构发生改变从而呈现不同颜色。	以酚红为例：阴：红阳：黄	直观、快速、简便	易受到样品干扰；非特异性检测
序列特异性的可视化检测方法
酶促底物显色法	HRP或G-四链体/血红素在H₂O₂的作用下催化显色底物显色	以ABTS^2-为例阴：无色阳：绿色	酶催化具有高效性、G-四链体DNAzyme成本低、	天然酶对环境要求高，DNAzyme催化活性会出现下降
纳米颗粒显色法	通过诱导AuNPs呈现不同状态(聚集/分散)，从而观察到颜色的转变	分散：红色聚集：紫色/离心后沉淀	直观、快速、低成本	难以实现单管多重检测、AuNPs状态易受体系影响
横向流动分析法	抗原抗体结合	阴：C线阳：T线&C线	直观、快速、可多重检测	脱靶现象；检测的灵活度受限

可视化分析在病原体核酸现场快速检测中的研究进展

Research progress of visual detection in rapid on-site detection of pathogen nucleic acid

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