Research Progress of Pathogen Detection Technologies Based on CRISPR/CAS Biosensor

doi:10.13523/j.cb.2105006

China Biotechnology

2021, Vol. 41

Issue (8): 67-74 DOI: 10.13523/j.cb.2105006

Research Progress of Pathogen Detection Technologies Based on CRISPR/CAS Biosensor

XU Wen-juan¹,SONG Dan¹,CHEN Dan¹,LONG Hui²,CHEN Yu-bao³,LONG Feng^1,^**(

)

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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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

Received: 06 May 2021 Published: 31 August 2021

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Corresponding Authors: Feng LONG E-mail: longf04@ruc.edu.cn

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Cite this article:

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

Fig.1 Scheme of CRISPR/Cas biosensing principle based on different Cas protein

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]

Table 1 Comparison of detection technologies for pathogenic bacteria based on CRISPR/Cas12

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]

Table 2 Comparison of detection technologies for pathogenic bacteria based on CRISPR/Cas13


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