Investigating enzyme kinetics and fluorescence sensing strategy of CRISPR/Cas12a for foodborne pathogenic bacteria.

Foodborne pathogenic bacteria are widespread in various foods, whose cross-contamination and re-contamination are critical influences on food safety. Rapid, accurate, and sensitive detection of foodborne pathogenic bacteria remains a topic of concern. CRISPR/Cas12a can recognize double-stranded DNA directly, showing great potential in nucleic acid detection. However, few studies have investigated the cleavage properties of CRISPR/Cas12a. In this study, the trans-cleavage properties of LbCas12a and AsCas12a were investigated to construct the detection methods for foodborne pathogenic bacteria. The highly sensitive fluorescent strategies for foodborne pathogens were constructed by analyzing the cleavage rates and properties of substrates at different substrate concentrations. Cas12a was activated in the presence of foodborne pathogenic target sequence was present, resulting in the cleavage of a single-stranded reporter ssDNA co-labelled by fluorescein quencher and fluorescein. The sensitivity and specificity of the Cas12a fluorescent strategy was investigated with Salmonella and Staphylococcus aureus as examples. The results showed that AsCas12a was slightly more capable of trans-cleavage than LbCas12a. The detection limits of AsCas12a for Salmonella and Staphylococcus aureus were 24.9 CFU mL-1 and 1.50 CFU mL-1, respectively. In all the seven bacteria, Staphylococcus aureus and Salmonella were accurately discriminated. The study provided a basis for constructing and improving the CRISPR/Cas12a fluorescence strategies. The AsCas12a-based detection strategy is expected to be a promising method for field detection.

A Simplified Amplification-Free Strategy with Lyophilized CRISPR-CcrRNA System for Drug-Resistant Salmonella Detection.

Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drug-resistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug-resistant Salmonella has an ultra-sensitive detection limit of 84 CFU mL-1 within 30 min, with good linearity in the range of 102 -106 CFU mL-1 . In real-world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC-TRFIA offers a cost-effective, sensitive, and virtually equipment-independent platform for preventing foodborne illnesses, screening for drug-resistant Salmonella, and guiding clinical use.

A Strategy for Sample Preparation: Using Egg White Gel to Promote the Determination of Aflatoxin M1 Content in Milk Samples.

The analysis of food samples is a challenging task. The high complexity of food matrices hinders the extraction and detection of analytes from them. Therefore, the correct preparation of food samples is a crucial step for their subsequent analysis, as it achieves the proper isolation and preconcentration of analytes and removes the interfering proportion of the food matrix before instrumental analysis. We aimed to develop a method that not only satisfies the requirement of detecting trace compounds in complex matrices but also achieves a "greener" approach by reducing the use of organic solvents and non-degradable materials to minimize the health hazards posed to the operators as well as pollution to the environment. In this study, we prepared egg white as a concentrated gel and used this material for the biological purification of milk samples. After the milk protein was removed by acidification and salting, the residual amount of aflatoxin M1 in milk samples was quantitatively determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the novel egg white purification method possessed advantages over the immunoaffinity technique used as the reference method in extraction recovery, sensitivity, repeatability, and operability. The limit of detection (LOD) was 0.001 µg/kg. In spiked samples containing 0.01 µg/kg to 2 µg/kg of AFM1, the average recovery was 88.3-94.7%, with a precision of 6.1-11.0%. Improved repeatability was obtained by significantly reducing the operation time and resource requirements compared with the immunoaffinity technique currently used internationally. This study provides a reference for the further improvement of the relevant international standards in place for the detection of aflatoxin M1 in milk.

A rapid and ultrasensitive dual detection platform based on Cas12a for simultaneous detection of virulence and resistance genes of drug-resistant Salmonella.

Accurate, sensitive, and rapid detection of Salmonella and determination of whether it carries drug resistance genes plays an important role in guiding the clinical medication of salmonellosis and laying a foundation for studying the mechanism of drug resistance transmission of Salmonella. Here, a novel nontransferable, ultrasensitive dual detection platform (Cas12a-Ddp) was developed. The round cap allowed for temporary storage of more Cas12a detection solution than flat cap, enabling one-pot assays and reducing aerosol contamination. The results were read out in dual mode by the microplate reader and UV visualization to achieve sensitive dual-target detection of the virulence genes and drug resistance genes of Salmonella simultaneously, with the possibility of onsite detection. Cas12a-Ddp was combined with multiple polymerase chain reactions and recombinase polymerase amplifications successively. An ultrasensitive dual detection limit of 1 CFU/mL was obtained without any cross-reaction within 40 min. This was an improvement of 1-2 orders of magnitude over the existing methods. Cas12a-Ddp overcame the influence of proteins and fat in liquid matrix foods. It was used for the detection of drug-resistant Salmonella in milk and skim milk powder, also with the dual detection limit of 1 CFU/mL and spiked recovery of 68.58%-158.49%. It was also used for the analysis of Salmonella resistance rate analysis. The Cas12a-Ddp provided a reliable, fast, sensitive, and practical multi-CRISPR detection platform.

One-step time-resolved fluorescence microsphere immunochromatographic test strip for quantitative and simultaneous detection of DON and ZEN.

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins that contaminate a wide range of grains and crops. In this study, a one-step time-resolved single-channel immunochromatographic test strip based on europium ion polystyrene fluorescence microspheres was first developed for sensitive and quantitative detection of DON and ZEN. The concentration of the artificial antigen and the mass ratio of the monoclonal antibody to fluorescent microspheres for conjugation were optimized to simplify the sample addition process during immunochromatographic assay and improve the on-site detection efficiency. The limits of detection (LOD) of the single-channel immunochromatographic test strip for DON and ZEN detection were 0.17 and 0.54 µg/L, respectively. Meanwhile, the dual-channel immunochromatographic test strip was designed to simultaneously detect DON and ZEN, with LODs of 0.24 and 0.69 µg/L achieved for DON and ZEN, respectively. The developed test strips also yielded recovery results consistent with that obtained by LC-MS/MS for DON and ZEN detection in real samples of wheat and corn flour, confirming the practicability and reliability of the test strip. The developed immunochromatographic test strips realize quick and sensitive detection of DON and ZEN, exhibiting potential for broad applications in the point-of-care testing platform of multiple mycotoxins in agricultural products. Graphic abstract.