Development and Validation of a CRISPR-Cas12a Enhanced PTS for Sensitive and Specific Detection of Aflatoxin B1 in Food Samples

Aflatoxin B1 (AFB1) is highly toxic and cause profound hepatotoxic, nephrotoxic, immunotoxic, mutagenic, teratogenic, and carcinogenic effects. It poses a severe risk to human health, particularly in developing countries where exposure is often chronic and uncontrolled, affecting billions of people globally. The robustness of AFB1, combined with its prevalence in staple crops like peanuts and maize, highlights the urgent need to develop efficient, accurate, and accessible detection methods to adhere to stringent global food safety standards. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, particularly the Cas12a enzyme, is an innovative detection method that has advanced genomic editing and, more recently, have been applied in diagnostic platforms due to their high specificity and sensitivity. To this end, a new study published in Chemical Communication and conducted by Ziqiang Deng, Jin Zhou, Chaoqun Wang, Jianyu Hu, Professor Rui Liu, and Professor Yi Lv at the Sichuan University, the authors developed a new modified pregnancy test strip (PTS)-CRISPR system for detecting AFB1 and demonstrated the effectiveness of this innovative food safety monitoring method.

The researchers engineered an AFB1-specific aptamer and coupled it with a reporter molecule. This complex was designed to bind AFB1, initiating a conformational change that activates Cas12a trans-cleavage activity. Upon AFB1 binding, the free aptamer triggered the CRISPR-Cas12a complex, which then cleaved a non-target DNA probe tagged with a hCG signal. This cleavage resulted in the accumulation of visible signals on commercial PTS. The authors showed that the assay successfully demonstrated that in the presence of AFB1, the Cas12a enzyme was activated, leading to a visible line on the PTS. The intensity of this line correlated with the concentration of AFB1, indicating a successful transduction of molecular recognition into a detectable visual output.

The team determined the sensitivity and quantitative capabilities of the PTS-CRISPR assay using different concentrations of AFB1 by preparing different known concentrations of AFB1 to test the assay’s sensitivity. Afterward the color intensity on the PTS was digitized using smartphone cameras and analyzed with image processing software (Image J) to quantify the AFB1 concentration. Indeed, the PTS-CRISPR assay’s design to incorporate the use of smartphones for quantifying results, is critical step towards digital integration and data management in food safety monitoring. By utilizing smartphones, which are widely accessible globally, the intensity of the test strip’s color can be digitally analyzed and quantified, allowing for precise and traceable measurements. This feature not only enhances the assay’s utility but also facilitates data sharing and real-time monitoring of food safety across different locations. It is worth mentioning that the assay exhibited a linear response to varying concentrations of AFB1, with a limit of detection (LOD) as low as 2.6 pg/mL. The results highlighted the assay’s potential for both qualitative and quantitative analysis, suitable for meeting stringent food safety standards.

The authors also tested the selectivity of the assay against potential interfering substances like other mycotoxins. To do this, other mycotoxins such as ochratoxin A (OTA), zearalenone (ZAE), and fumonisin B1 (FB1) were tested alongside AFB1 to evaluate the specificity. They also introduced these mycotoxins into the assay at concentrations 10 times higher than that of AFB1 to perform competitive assay. The assay showed high specificity for AFB1, with negligible cross-reactivity from other mycotoxins. This demonstrated the system’s robustness against interference, confirming its suitability for AFB1 detection without false positives from similar substances. Moreover, to validate the PTS-CRISPR method in real-world scenarios by detecting AFB1 in contaminated agricultural products, they chose two common crops susceptible to AFB1 contamination, maize and peanuts. Maize flour and peanut oil were used as sample matrices. They spiked known amounts of AFB1 into these samples, and the recovery rate of the assay was measured to assess accuracy. The authors demonstrated the recovery rates ranged from 84.98% to 98.11%, with relative standard deviations (RSDs) between 3% and 22%. These results indicated that the PTS-CRISPR assay could reliably detect AFB1 in complex food matrices, although variations suggested that matrix effects could impact assay performance.

The authors’ findings are significant because the study successfully integrates the CRISPR technology, specifically the Cas12a enzyme, into a point-of-care testing format using a commercial PTS. This novel approach brings the high specificity and sensitivity of CRISPR-based genetic editing tools into the realm of food safety, making it a pioneering effort in this field. Moreover, the application of CRISPR technology in a low-cost, easy-to-use pregnancy test format addresses significant barriers in food safety monitoring, particularly in resource-limited settings. By lowering the cost and technical requirements, this method democratizes access to advanced biotechnological tools, enabling broader implementation across various global regions, especially in developing countries where AFB1 contamination is prevalent and often unmonitored. Furthermore, the PTS-CRISPR system allows for the rapid detection of AFB1 directly at the point of need, without requiring samples to be sent to a laboratory. This on-site testing capability is crucial for timely decisions regarding the safety of food products, potentially preventing the consumption of contaminated food and reducing the risk of exposure to AFB1’s severe health effects. Additionally, while traditional point of care systems, such as PTS for pregnancy, generally offer qualitative results, the PTS-CRISPR system developed in this study provides quantitative data on AFB1 concentrations. This capability is enhanced by the use of smartphone integration for reading and analyzing results, which adds a layer of precision and data management previously unavailable in such low-cost formats.

Indeed, the modular nature of the CRISPR system and the general approach of coupling it with a PTS suggest that this technology could be adapted to detect a wide range of other contaminants and pathogens, beyond just AFB1. This flexibility holds significant potential for expanding the scope of rapid and affordable point of care across various domains of public health and environmental monitoring. The study contributes directly to efforts aimed at safeguarding public health by providing a means to enforce food safety standards more effectively. The ability to detect AFB1 efficiently and affordably helps in aligning with international food safety regulations, thereby enhancing the global trade compliance of food products from regions with high risks of AFB1 contamination.  Overall, the PTS-CRISPR method developed by the Sichuan University researchers proved to be a highly effective tool for the rapid, sensitive, and specific detection of AFB1. The method’s low cost, ease of use, and rapid turnaround make it an appealing option for on-site food safety testing, particularly in resource-limited settings. The successful application of this technology to real food samples further demonstrates its potential for widespread use in global food safety monitoring, aligning with the critical need for innovative solutions in the fight against foodborne contaminants.