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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (8): 67-74    DOI: 10.13523/j.cb.2105006
综述     
基于CRISPR/Cas生物传感原理的病原菌检测技术研究进展*
徐文娟1,宋丹1,陈丹1,龙辉2,陈禹保3,龙峰1,**()
1 中国人民大学环境学院 北京 100875
2 广西壮族自治区产品质量检验研究院 南宁 530200
3 中国医学科学院医学实验动物研究所 北京 100021
Research Progress of Pathogen Detection Technologies Based on CRISPR/CAS Biosensor
XU Wen-juan1,SONG Dan1,CHEN Dan1,LONG Hui2,CHEN Yu-bao3,LONG Feng1,**()
1 School of Environmental & Natural Resources, Renmin University, Beijing 100875, China
2 Institute of Product Quality Inspection, Guangxi Zhuang Autonomous Region, Nanning 530007, China
3 Institute of Laboratory Animal Sciences, Beijing　100021, China
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摘要：

病原菌的快速准确检测是实现疫情高效防控、疾病精准治疗、污染环境及时处置的关键。而现有的病原菌现场快速检测技术,主要以定性分析为主,假阳性/假阴性受到诟病,检测准确性仍有待提升,亟待发展基于新原理、新方法的病原菌快速检测技术。基于CRISPR(clustered regularly interspaced short palindromic repeats)的生物传感技术因具有高灵活性(对不同的基因靶点只需改变crRNA序列)、高特异性(单碱基分辨)、高灵敏(优于10-18 mol/L浓度)、可编程、可模块化、低成本、可在各种体外介质中高效稳定运行等独特优势,打破了传统分子诊断与检测技术的局限性,正在成为下一代病原菌检测技术的引领者。在该技术中,Cas效应蛋白被用作高特异性的序列识别元件,结合不同的生物传感机制,即可用于病原菌的高特异性快速灵敏检测。在总结CRISPR/Cas生物传感技术原理的基础上,综述了用于病原菌检测的CRISPR/Cas12和CRISPR/Cas13生物传感技术研究进展。通过阐述CRISPR/Cas生物传感技术在实际应用中面临的挑战,展望其未来的发展前景。
关键词： 病原菌;  规律间隔成簇短回文重复序列;  生物传感器;  脱氧核糖核酸;  核糖核酸    
Abstract:

Rapid and accurate detection of pathogens is essential to achieve efficient epidemic prevention and control, accurate treatment of diseases, and timely disposal of polluted environment. However, the existing on-site rapid detection methods of pathogenic bacteria mainly focuses on qualitative analysis. False positive/negative results exist and their detection accuracy still needs to be improved. It is urgent to develop rapid detection technologies of pathogenic bacteria by taking use of new principles and methods. The CRISPR (clustered regularly interspaced short palindromic repeats) based biosensors have several unique advantages, such as high flexibility (only needing to change the crRNA sequence for different target genes), high specificity (single base resolution), high sensitivity (better than 10-18 mol/L concentration), programmability, modularity, low cost, and high efficiency and stability in various in vitro media. It has become the leader of the next generation of pathogen detection technologies without the limitations of traditional molecular diagnosis and detection technologies. In this technology, Cas effector proteins are used as highly specific sequence recognition elements. Through combined with various biosensor mechanisms, they can be used for rapid and sensitive detection of pathogens with high specificity. After summarizing the principle of the CRISPR/Cas biosensor technology, the research progress of the CRISPR/Cas12 and CRISPR/Cas13 biosensor technologies for pathogen detection was reviewed. The challenges of the CRISPR/Cas biosensor technology in practical application are discussed, and its future developments are prospected.
Key words: Pathogenic bacteria    Clustered regularly interspaced short palindromic repeats    Biosensor    DNA RNA
收稿日期: 2021-05-06 出版日期: 2021-08-31
ZTFLH:  Q812  
基金资助: * 国家自然科学基金资助项目(21077063)
通讯作者: 龙峰     E-mail: longf04@ruc.edu.cn
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引用本文:

徐文娟,宋丹,陈丹,龙辉,陈禹保,龙峰. 基于CRISPR/Cas生物传感原理的病原菌检测技术研究进展*[J]. 中国生物工程杂志, 2021, 41(8): 67-74.

XU Wen-juan,SONG Dan,CHEN Dan,LONG Hui,CHEN Yu-bao,LONG Feng. Research Progress of Pathogen Detection Technologies Based on CRISPR/CAS Biosensor. China Biotechnology, 2021, 41(8): 67-74.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105006    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I8/67

图1  基于不同效应蛋白的CRISPR/Cas生物传感原理示意图[3]
Cas 12
类型		 病原菌 核酸 系统名 信号放大方式 灵敏度 检测方式 检测时间 样品
处理		 参考
文献
Cas12a SARS-CoV-2 RNA OR-SHERLOCK RT-RPA 2.5 copies/μL 荧光检测 50 min 需要 [25]
SARS-CoV-2 RNA CRISPR-Cas12a LAMP 20 copies/μL 裸眼 40 min 否 [26]
SARS-CoV-2 RNA CRISPR-Cas12a RPA 约5 copies/μL 裸眼 20 min - [8]
SARS-CoV-2 RNA CODA RT-RPA 3 copies/μL 荧光检测 20 min 否 [27]
SARS-CoV-2 RNA opvCRISPR RT-LAMP 5 copies 裸眼 45 min 否 [28]
SARS-CoV-2 RNA CRISPR-FDS RT-RPA 5 copies 荧光检测 50 min [29]
人乳头瘤病毒16型
(HPV16)和细小
病毒B19型(PB19)		 DNA E-CRISPR - 1 pmol/L 电化学 30 min 需要 [30]
乙型脑炎病毒(JVE)
和伪狂犬病毒
(PRV)		 DNA/
RNA		 HOLMES PCR 10 amol/L 荧光检测 1 h 需要 [20]
疟原虫(Plasmodium) DNA SHERLOCK+
CRISPR		 RPA 5 amol/L 荧光检测 1 h 需要 [31]
甲型和乙型流感病毒
(Influenza A and B virus)		 RNA CRISPR-Cas12a RT-RPA+RT-
LAMP		 1×100 PFUs 荧光检测/
侧向流动检测		 约1.5 h 需要 [32]
非洲猪瘟(ASF) DNA CRISPR-Cas12a PCR/LAMP 2 copies/μL 裸眼 40 min 需要 [33]
SARS-CoV-2 RNA ENHANCE RT-LAMP N基因0.2
copies/μL,E基
因7.9 copies/μL		 荧光检测/
侧向流动检测		 30 min 需要 [34]
SARS-CoV-2 RNA RT-LAMP+
Cas12a		 RT-LAMP 20 copies/μL 侧向流动检测 <40 min 需要 [16]
SARS-CoV-2 RNA CRISPR Cas12a RT-RAA 10 copies/μL 裸眼 <45 min 需要 [35]
非洲猪瘟(ASF) DNA CORDS RAA 1 fmol/L 荧光检测/
侧向流动检测		 1 h 需要 [36]
非洲猪瘟(ASF) DNA POC RPA/LAMP 100 fmol/L 荧光检测 <2 h 需要 [37]
非洲猪瘟(ASF) DNA CRISPR Cas
colorimetric		 RPA 200 copies/μL 裸眼 <1 h 需要 [38]
伪狂犬病毒(PRV) RNA HOLMES RT-PCR 1~10 amol/L 荧光检测 约1 h 需要 [39]
金黄色葡萄球菌
(Staphylococcus aureus)		 DNA CRISPR/Cas12a+
logic gates		 PCR 103 CFU/mL 荧光检测 2 h 需要 [40]
Cas 12
类型		 病原菌 核酸 系统名 信号放大方式 灵敏度 检测方式 检测时间 样品
处理		 参考
文献
人乳头瘤病毒16/18型
(HPV16/18)		 DNA DETECTR RPA 10 pmol/L 荧光检测 1 h 需要 [19]
SARS-CoV-2/艾滋
病1型(HIV-1)		 RNA AIOD-CRISPR RPA 11 copies/μL 荧光检测/
裸眼		 20 min - [41]
SARS-CoV-2 RNA STOPCovid RT-LAMP 100 copies 荧光检测/
侧向流动检测		 55 min/
95 min		 需要 [42]
Cas12b 乙型脑炎病毒(JVE) DNA/
RNA		 HOLMESv2 LAMP 10 amol/L 荧光检测 1 h - [22]
SARS-CoV-2 RNA CASdetec RT-RAA 1×104 copies/mL 裸眼 约50 min 需要 [43]
人乳头瘤病毒16/18型
(HPV16/18)		 DNA CDetection RPA 1 amol/L 荧光检测 约3 h 需要 [39]
表1  基于CRISPR/Cas12的病原菌检测技术比较
Cas 13
类型		 病原菌 核酸 系统名 信号放大方式 灵敏度 检测方式 检测
时间		 样品
处理		 参考
文献
Cas 13a 蜡状芽孢杆菌
(Bacillus cereus)		 RNA Light-up RNA
aptamer signaling-
CRISPR-Cas13a		 - 10 CFU 荧光检测 20 min 需要 [47]
寨卡病毒(Zika)和登革
热病毒(Dengue virus)		 RNA CRISPR-Dx RT-RPA 1.25 copies/μL 荧光检测 约20 min 需要 [12]
埃博拉病毒(Ebola virus) RNA SHERLOCK - 20 pfu/μL 荧光检测 <1 h 需要 [48]
新冠病毒(SARS-CoV-2) RNA CRISPR-Cas13a - 100 copies/μL 手机相机 30 min - [49]
RNA SHINE RPA 10 copies/μL 手机软件 <1 h 需要 [50]
寨卡病毒(Zika)或登革
热病毒(Dengue virus)		 RNA SHERLOCK LAMP 2 amol/L 荧光检测/
侧向流动检测		 0.5~3h 需要 [11]
金黄色葡萄球菌
(Staphylococcus aureus)		 DNA CCB-Detection PCR 1 amol/L 荧光检测 <4 h 需要 [51]
H7N9型禽流感 RNA CRISPR-Cas13a RT-RPA 1 fmol/L 荧光检测 50min 需要 [52]
犬细小病毒2型(CPV-2) DNA SHERLOCK RPA 100 amol/L 荧光检测 <0.5h 需要 [53]
寨卡病毒(Zika)和登革
热病毒(Dengue virus)		 RNA HUDSON+
SHERLOCK		 RT-RPA 1 copies/μL 荧光检测/
侧向流动检测		 <1 h 否 [23]
人类疱疹病毒(EBV) DNA SHERLOCK RPA 8 copies qPCR装置 <2 h 需要 [54]
埃博拉病毒(Ebola virus) RNA CRISPR-Cas13a RT-PCR 20 pfu / mL 荧光检测 <5 min 需要 [48]
淋巴细胞性脉络膜脑膜
炎病毒(LCMV)		 RNA SHERLOCK RT-RPA amol/L 荧光检测 约2h 需要 [55]
蓝耳病病毒(PRRSV) RNA CRISPR-Cas13a RT-RPA 172 copies/μL 荧光检测/
侧向流动检测		 1~3 h 均可 [56]
副溶血性弧菌(Vibrio
parahemolyticus)		 DNA CRISPR-Cas13a RAA 10 copies/反应 荧光检测 1 h 需要 [57]
埃博拉病毒(Ebola virus)
和拉萨病毒(Lassa virus)		 RNA SHERLOCK+
SUDSON		 RPA 10 copies /μL 荧光检测 1~3 h 否 [58]
Cas 13b 甲型流感病毒(IAV)和
水泡性口炎病毒(VSV)		 RNA SHERLOCK RT-RPA amol/L 荧光检测 约2h 需要 [55]
表2  基于CRISPR/Cas13的病原菌检测技术比较
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