An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection

Li, Fan; Ye, Qinghua; Chen, Moutong; Zhou, Baoqing; Zhang, Jumei; Pang, Rui; Xue, L.; Wang, Juan; Zeng, Haiyan; Wu, Shi; Zhang, Youxiong; Ding, Yu; Wu, Qingping

doi:10.1016/j.bios.2021.113073

Cited by 179 publications

(95 citation statements)

References 32 publications

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“…The most widely used hybridization chain reaction (HCR) is a simple, enzyme-free, robust, and efficient isothermal amplification of nucleic acids; long, linear duplex concatamers are formed through the cross-opening and self-assembling of two DNA hairpins initiating by a target probe [ 7 , 8 ]. In addition, target-free HCR amplification is a probe amplification technique; cross-contamination and false-positive results frequently occurring in LAMP, RPA, and PCR are effectively reduced [ 17 ]. To date, HCR has been explored to detect a variety of targets, such as nucleic acids, proteins, and pathogens [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…Combining Cas12a with HCR has provided a detection method for targets [ 10 ]. Based on HCR and CRISPR-Cas12a, Xing et al developed an aptamer-HCR-CRISPR-Cas12a fluorescence readout approach for the direct detection of tumor-derived extracellular vesicle proteins [ 17 ]. Kachwala combined HCR with CRISPR-Cas12a to develop a gel electrophoresis-based assay.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we intended to introduce HCR-based CRISPR-Cas12a into an electrochemical biosensor platform for sensitive and specific detection of S. typhimurium (Scheme 1 ) [ 21 ] Electrochemical biosensors have been widely applied to detect pathogenic bacteria in food safety processes due to their fast signal readouts, simplicity, high sensitivity, and low cost, thus providing a practical or deployable point-of-care system [ 17 ]. The nonspecific ssDNA reporter (methylene blue (MB) tag modified on hairpin DNA (hpDNA)) was drafted with an MB electrochemical tag for signal transduction and a thiol moiety to tether on the sensor surface to acquire the signal electrically.…”

Section: Introductionmentioning

confidence: 99%

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Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

Liu

Feng

et al. 2021

Anal Bioanal Chem

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In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium . Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03733-6.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

Liu

Feng

et al. 2021

Anal Bioanal Chem

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“…The Cas nucleases contain RNA-guided RNases (Cas13a and Cas13b) and RNA-guided DNases (Cas12a, Cas12b and Cas14), which are able to exhibit non-specific trans-cleavage activity after binding to their specific targets and has been fully evaluated for the detection of nucleic acids [ 26 , 27 , 28 ]. For Cas orthologues, Cas12a and Cas13 are the main nucleases for the development of CRISPR-Cas-based nucleic acid detection method with higher sensitivity and specificity for the capability of recognizing a nucleic acid target, and they also have been developed to combine with isothermal amplification technologies, such as Recombinase-aided Amplification (RAA), to enhance the diagnostic accuracy [ 29 , 30 , 31 ]. Depending on the type of nucleic acid substrate (DNA or RNA), CRISPR-Cas12a/Cas13-based systems have been alternatively applied to the detection of different pathogens, including viruses [ 26 , 29 , 32 , 33 , 34 ], bacteria [ 31 , 35 , 36 , 37 ], parasitic protozoa Plasmodium spp.…”

Section: Introductionmentioning

confidence: 99%

RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA)

Wang

Zheng

et al. 2021

Microorganisms

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Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.

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“…20 On the basis of this discovery, they reported and applied Cas12a protein by combining recombinase polymerase amplification (RPA) to develop a rapid and accurate test for the detection and classification of human papillomavirus (HPV) in clinical specimens. 20 Based on the ssDNase property of the Cas12a protein, it expands upon the diagnostic applications range of infectious and noninfectious diseases in various situations, such as the detection of virus (i. e. pandemic COVID-19 21 , SARS-CoV-2 22 , and African swine fever virus 23 ) and bacteria 24 (i. e. Staphylococcus aureus, 25 Listeria monocytogenes, 26 Mycobacterium tuberculosis 27 , and Methicillin-resistant Staphylococcus aureus (MRSA) 28 ), it can also be used for pathogen detection in agricultural 29 and aquatic community. 30 CRISPR technology is expected to become a rapid, accurate and portable diagnostic tool in the future.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes

Wang

Cai

et al. 2021

Preprint

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Background: Dermatophytosis is an infectious disease of global significance caused by several fungal species, which affects the hair, nails, or superficial layers of the skin. The most common zoonotic dermatophytes are Microsporum canis, Nannizzia gypsea and Trichophyton mentagrophytes. Wood's lamp examination, microscopic identification and fungal culture are the main conventional diagnostic methods used in clinics. Less common methods are dermatophyte PCR and biopsy/histopathology. However, these methods also have limitations for providing both accuracy and timely on-site detection. The recent development of CRISPR-based diagnostic platform provides the possibility of a rapid, accurate, and portable diagnostic tool, which has huge potential for clinical applications. Objectives: The purpose of this study is to establish a molecular method for rapid and accurate diagnosis of clinical dermatophytes, which can accelerate clinical diagnostic testing and help timely treatment. Methods: In this paper, we design a Cas12a-based assay combined with recombinase polymerase amplification (RPA) to differentiate three main zoonotic dermatophytes. The limit of detection (LOD) is determined by using standard strains. A total of 25 clinical samples (hair and scurf) are identified to evaluate the sensitivity and specificity of this assay. Results: The RPA-Cas12a method showed high sensitivity and specificity (100% and 100%, respectively). The results could be observed directly by naked-eyes, and all tested samples were consistent with fungal culture and sequencing results. Conclusions: Compared with other methods, the RPA-Cas12a-fluorescence assay requires less time (30 minutes) and less complicated equipment, and visible changes can be clearly observed, which is suitable for on-site clinical diagnosis.

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An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection

Cited by 179 publications

References 32 publications

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA)

Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes

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