An electrochemical sensing method based on CRISPR/Cas12a system and hairpin DNA probe for rapid and sensitive detection of Salmonella Typhimurium

He, Yawen; Jia, Fei; Sun, Yanchao; Fang, Weihuan; Li, Yanbin; Chen, Juhong; Fu, Yingchun

doi:10.1016/j.snb.2022.132301

Cited by 21 publications

(7 citation statements)

References 26 publications

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“…Compared with the results of gel electrophoresis and qPCR methods (with LOD of 8.1 × 10 3 and 8.1 × 10 2 CFU/mL, respectively, in Figure S3 ), the proposed dual-mode PCR biosensor showed superior performance. Furthermore, it exhibited comparable or even higher sensitivity, a relatively wide detection range, and a short detection time compared with the analytical performance of other reported methods ( Table 1 ) [ 37 , 38 , 39 , 40 , 41 , 42 ], confirming its ideal performance.…”

Section: Resultssupporting

confidence: 58%

Development of a Dual Mode UCNPs-MB Biosensor in Combination with PCR for Sensitive Detection of Salmonella

et al. 2023

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In recent years, the high prevalence of Salmonella has emerged as a serious threat to public safety, prompting attempts to utilize accurate, rapid, and direct methods to ensure food safety. In this study, a multifunctional platform featuring dual-mode detection channels (colorimetric-fluorescence) combined with polymer chain reaction (PCR) was proposed for the sensitive and rapid detection of Salmonella. Additionally, the colorimetric measurements were achieved by color changes induced by methylene blue (MB) insertion into the double-stranded DNA, and the fluorescence measurements were performed by internal filter effect (IFE)-induced fluorescence quenching of upconversion nanoparticles (UCNPs) by MB. The results showed that the IFE and PCR amplification processes improved the sensitivity of the sensor towards Salmonella detection, with a limit of detection (LOD) of 21.8 CFU/mL. Moreover, this colorimetric-fluorescence dual-mode PCR biosensor was applied to determine Salmonella in food samples, such as chicken, egg, and fish, which produced satisfactory results. Overall, the present study results demonstrate the potential for combining PCR amplification with IFE to develop an efficient and reliable dual-mode analysis platform to safeguard food security.

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Section: Resultssupporting

confidence: 58%

Development of a Dual Mode UCNPs-MB Biosensor in Combination with PCR for Sensitive Detection of Salmonella

et al. 2023

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“…aureus SEA recombinant plasmid ranging from 0 to 10 6 CFU/mL were prepared and subjected to LAMP, followed by CAMURE detection. We realized that the sensitivity of CAMURE-LAMP was boosted to 10 CFU/mL (Figures d–f and S2), which is comparable to other CRISPR/Cas system-coupled amplification techniques. ,− However, CAMURE is versatile in a variety of readouts, more practical with minimum reagents consumption, and less laborious for results validation.…”

Section: Resultsmentioning

confidence: 70%

A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts

et al. 2023

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The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in endpoint readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2−5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.

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“…CrRNA is a crucial component of the CRISPR/Cas detection system, as it induces subsequent enzyme cleavage through its specific targeting behavior. , Furthermore, due to its sequence-specific recognition of target nucleic acids, it determines the specificity of CRISPR-based detection. Thus, the crRNA design is important, and target DNA sequences should contain protospacer adjacent motif sites rich in T .…”

Section: Resultsmentioning

confidence: 99%

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification

Chen,

Zhao,

Han

et al. 2023

J. Agric. Food Chem.

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A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.

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An electrochemical sensing method based on CRISPR/Cas12a system and hairpin DNA probe for rapid and sensitive detection of Salmonella Typhimurium

Cited by 21 publications

References 26 publications

Development of a Dual Mode UCNPs-MB Biosensor in Combination with PCR for Sensitive Detection of Salmonella

Development of a Dual Mode UCNPs-MB Biosensor in Combination with PCR for Sensitive Detection of Salmonella

A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification

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