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基于 RAA-CRISPR/Cas12a 系统的 O157:H7 的超灵敏和快速可视化检测

Ultrasensitive and Rapid Visual Detection of O157:H7 Based on RAA-CRISPR/Cas12a System.

机构信息

Institute of Environmental Research at Greater Bay Area, Key Laboratory for Water Quality and Conservation of the Pearl River Delta, Ministry of Education, Guangzhou University, Guangzhou 510006, China.

Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, School of Biomedical Engineering, Shenzhen Campus of Sun Yat-Sen University, Shenzhen 518107, China.

出版信息

Biosensors (Basel). 2023 Jun 16;13(6):659. doi: 10.3390/bios13060659.


DOI:10.3390/bios13060659
PMID:37367024
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10295930/
Abstract

() O157:H7 is a major foodborne and waterborne pathogen that can threaten human health. Due to its high toxicity at low concentrations, it is crucial to establish a time-saving and highly sensitive in situ detection method. Herein, we developed a rapid, ultrasensitive, and visualized method for detecting O157:H7 based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. The CRISPR/Cas12a-based system was pre-amplified using the RAA method, which showed high sensitivity and enabled detecting as low as 1 CFU/mL (fluorescence method) and 1 × 10 CFU/mL (lateral flow assay) of O157:H7, which was much lower than the detection limit of the traditional real-time PCR technology (10 CFU/mL) and ELISA (1010 CFU/mL). In addition, we demonstrated that this method still has good applicability in practical samples by simulating the detection in real milk and drinking water samples. Importantly, our RAA-CRISPR/Cas12a detection system could complete the overall process (including extraction, amplification, and detection) within 55 min under optimized conditions, which is faster than most other reported sensors, which take several hours to several days. The signal readout could also be visualized by fluorescence generated with a handheld UV lamp or a naked-eye-detected lateral flow assay depending on the DNA reporters used. Because of the advantages of being fast, having high sensitivity, and not requiring sophisticated equipment, this method has a promising application prospect for in situ detection of trace amounts of pathogens.

摘要

O157:H7 是一种主要的食源性和水源性致病菌,可能会威胁人类健康。由于其在低浓度下具有高毒性,因此建立一种省时且高度敏感的原位检测方法至关重要。在此,我们开发了一种基于重组酶辅助扩增(RAA)和 CRISPR/Cas12a 技术的快速、超灵敏和可视化检测 O157:H7 的方法。该基于 CRISPR/Cas12a 的系统通过 RAA 方法进行预扩增,表现出高灵敏度,能够检测低至约 1 CFU/mL(荧光法)和 1×10 CFU/mL(侧流法)的 O157:H7,比传统实时 PCR 技术(10 CFU/mL)和 ELISA(10~10 CFU/mL)的检测限低得多。此外,我们通过模拟实际牛奶和饮用水样本中的检测,证明了该方法在实际样本中仍具有良好的适用性。重要的是,在优化条件下,我们的 RAA-CRISPR/Cas12a 检测系统可以在 55 分钟内完成整个过程(包括提取、扩增和检测),这比大多数其他报道的传感器快,大多数其他传感器需要几个小时到几天的时间。根据所使用的 DNA 报告分子,信号读出还可以通过手持式紫外灯产生的荧光或肉眼检测的侧流法进行可视化。由于该方法具有快速、高灵敏度且不需要复杂设备的优点,因此在痕量病原体的原位检测方面具有广阔的应用前景。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/019844d18a25/biosensors-13-00659-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/677b7791ae15/biosensors-13-00659-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/23ed8e963c21/biosensors-13-00659-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/9d854dbed5a4/biosensors-13-00659-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/15069949f994/biosensors-13-00659-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/019844d18a25/biosensors-13-00659-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/677b7791ae15/biosensors-13-00659-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/23ed8e963c21/biosensors-13-00659-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/9d854dbed5a4/biosensors-13-00659-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/15069949f994/biosensors-13-00659-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd1c/10295930/019844d18a25/biosensors-13-00659-g005.jpg

相似文献

[1]
Ultrasensitive and Rapid Visual Detection of O157:H7 Based on RAA-CRISPR/Cas12a System.

Biosensors (Basel). 2023-6-16

[2]
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J AOAC Int. 2022-12-22

[3]
Filtration-based LAMP-CRISPR/Cas12a system for the rapid, sensitive and visualized detection of Escherichia coli O157:H7.

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[4]
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[5]
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[6]
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Anal Chim Acta. 2023-1-25

[7]
The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7.

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[8]
Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.

J Food Prot. 2014-1

[9]
Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk.

J Dairy Sci. 2022-2

[10]
DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

Biosens Bioelectron. 2016-7-15

引用本文的文献

[1]
Biosensing Strategies to Monitor Contaminants and Additives on Fish, Meat, Poultry, and Related Products.

Biosensors (Basel). 2025-6-30

[2]
Biosensors for Monitoring, Detecting, and Tracking Dissemination of Poultry-Borne Bacterial Pathogens Along the Poultry Value Chain: A Review.

Animals (Basel). 2024-11-1

[3]
CRISPR-Cas target recognition for sensing viral and cancer biomarkers.

Nucleic Acids Res. 2024-9-23

[4]
Establishment of a Simple, Sensitive, and Specific Detection Method Based on Recombinase-Aided Amplification Combined with dsDNA-Specific Nucleases.

Foods. 2024-4-29

[5]
A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant .

Front Microbiol. 2024-3-6

[6]
Polyphenols and CRISPR as Quorum Quenching Agents in Antibiotic-Resistant Foodborne Human Pathogens ( Typhimurium, and 0157:H7).

Foods. 2024-2-15

[7]
An Emerging Foodborne Pathogen Spotlight: A Bibliometric Analysis and Scholarly Review of O157 Research.

Antibiotics (Basel). 2024-1-8

本文引用的文献

[1]
A Novel Hybrid Platform for Live/Dead Bacteria Accurate Sorting by On-Chip DEP Device.

Int J Mol Sci. 2023-4-11

[2]
Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.

Appl Biol Chem. 2023

[3]
Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters.

J Am Chem Soc. 2022-9-14

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Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.

Sci Total Environ. 2022-9-10

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Novel Micro-Nano Optoelectronic Biosensor for Label-Free Real-Time Biofilm Monitoring.

Biosensors (Basel). 2021-9-29

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LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus.

Transbound Emerg Dis. 2022-7

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J Pharm Biomed Anal. 2021-9-10

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Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP.

Sens Actuators B Chem. 2021-10-15

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Chem Sci. 2021-3-2

[10]
CRISPR/Cas12a-Assisted Ligation-Initiated Loop-Mediated Isothermal Amplification (CAL-LAMP) for Highly Specific Detection of microRNAs.

Anal Chem. 2021-6-8

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