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Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenicEscherichia colistrains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenicE. coliisolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes—stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.
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Ultrasensitive detection of acephate based on carbon quantum dot-mediated fluorescence inner filter effects
Acephate is an organophosphorus pesticide (OP) that is widely used to control insects in agricultural fields such as in vegetables and fruits. Toxic OPs can enter human and animal bodies and eventually lead to chronic or acute poisoning. However, traditional enzyme inhibition and colorimetric methods for OPs detection usually require complicated detection procedures and prolonged time and have low detection sensitivity. High-sensitivity monitoring of trace levels of acephate residues is of great significance to food safety and human health. Here, we developed a simple method for ultrasensitive quantitative detection of acephate based on the carbon quantum dot (CQD)-mediated fluorescence inner filter effect (IFE). In this method, the fluorescence from CQDs at 460 nm is quenched by 2,3-diaminophenazine (DAP) and the resulting fluorescence from DAP at 558 nm is through an IFE mechanism between CQDs and DAP, producing ratiometric responses. The ratiometric signal I 558 / I 460 was found to exhibit a linear relationship with the concentration of acephate. The detection limit of this method was 0.052 ppb, which is far lower than the standards for acephate from China and EU in food safety administration. The ratiometric fluorescence sensor was further validated by testing spiked samples of tap water and pear, indicating its great potential for sensitive detection of trace OPs in complex matrixes of real samples.
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- PAR ID:
- 10386183
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 147
- Issue:
- 23
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 5462 to 5469
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D2AN01552H
