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.

Carbon dots cooperatively modulating photocatalytic performance and surface charge of O-doped g-C3N4 for efficient water disinfection.

The emergence of widespread microbial contamination and drug-resistant bacteria in the water environment poses a severe threat to public health. Photocatalysis is considered an efficient, energy-saving, and cost-effective disinfection strategy for effectively removing microbial contamination from water bodies. In this paper, a metal-free O-doped g-C3N4/carbon dots (O-CN/CDs) nanosheet photocatalysts are prepared in coordination with various modification strategies, which results in a considerable improvement in photocatalytic disinfection activity compared to bulk g-C3N4 (B-CN). First, O-doping and morphology modulation are achieved simultaneously with hydrothermal treatment, which not only hinders the recombination of photogenerated hole-electron pairs, but also enables the exposure of more active centers. Subsequently, loading of CDs onto O-CN nanosheets by electrostatic self-assembly increases the production of photogenerated hole-electron pairs by expanding the visible light absorption region and promoted the separation of photogenerated carriers by trapping photogenerated electrons. Interestingly, the loading of CDs changes the charge on the surface of the composite photocatalyst from negative to positive, making it easier for the active species to come in contact with bacteria, and thus improving bacterial disinfection performance. Under visible light irradiation, the inactivation efficiency of optimized O-CN/CDs against methicillin-resistant Staphylococcus aureus (MRSA) is log(C/C0) = 4.08, approximately 9 times higher than that of B-CN. The main active species inactivating bacteria are superoxide anions radicals (•O2-) and photogenerated holes, and their attack causes damage to the membrane wall structure and leakage of intracellular components. Additionally, the feasibility of the as-prepared photocatalysts in water disinfection in real environments was confirmed by photocatalytic disinfection experiments in successive recovery cycles and in practical lake water.

First Report of Alternaria alternata Causing Leaf Spot on Bellis perennis in China.

Bellis perennis L., commonly known as the daisy or sun chrysanthemum, belonging to the family Asteraceae, is a perennial herb and is usually used as an ornamental plant worldwide for its vibrant flowers. Simultaneously, B. perennis has been proved to have therapeutic effects used on common colds, wound healing, anti-tumor, anxiolytic and antioxidant (Karakas et al. 2017). In July 2021, typical leaf spot was observed on B. perennis with about 50% disease incidence in Ruyue lake wetland park of Zibo (36.71°N, 118.01°E), Shandong Province, China. We surveyed more than 1000 square meters of planting area, and the diseased leaves were mostly concentrated in the lower location of plants, where the humidity was higher under the forest. Symptoms on the initially diseased leaves appeared as light yellow, round or oval lesions with light or brown borders. With the development of the disease, the area of the lesion gradually expands, the color deepens, and the shape is becoming irregular. To identify the causal pathogen, small pieces of 15 tissues collected from the infected leaves were sterilized with 75% ethanol for 30 s and then 2% sodium hypochlorite (NaClO) for 60 s, finally rinsed with sterile water three times. All the tissues were placed on potato dextrose agar (PDA) and incubated at 25 ℃ in the dark for 5 days (Zhu et al. 2013). A total of 13 isolates were obtained from the above diseased leaves. The cultures were initially grayish white, then a light green halo appeared in the middle of the medium after 5 days, with numerous gray aerial hyphae. For molecular identification, the RNA polymerase II beta subunit (PRB2), Tsr ribosome biogenesis protein, partial coding sequences of chitin synthase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and major allergen Alt a 1 were amplified from genomic DNA extracted from four representative single isolates using the primers PRB2DF/PRB2DR, Tsr1F/Tsr1R, CHSDF1/CHSDR1, GDF1/GDR1, and AltF/AltR (Damn et al. 2019; Lawrence et al. 2013), respectively, and sequenced (GenBank accession nos. OL416000, OL416001, OL416002, OL416003, and OL416004). These genes had more than 99.9% nucleotide identity with the corresponding sequences (KY131957.1, KY131958.1, KY996470.1, MN657411.1, and KY923227.1) of the reference strains of Alternaria alternata in GenBank. For pathogenicity tests, five healthy B. perennis plants each with three living leaves were inoculated with mycelial plugs of A. alternata from a 5-day-old culture grown on PDA. After inoculation, the plants were placed in a greenhouse with 85% relative humidity at 25 ℃ and monitored daily for symptom development. After 3 days, all inoculated leaves with mycelial plugs of A. alternata appeared symptoms similar to those observed in the field previously, while no symptoms appeared on negative controls which were inoculated with sterile PDA plugs. Cultures re-isolated from diseased leaves had the same morphological and molecular results as those isolated in the field, confirming Koch's postulates. The causal agent on B. perennis was confirmed as A. alternata on the basis of morphological and molecular results (Simmons 2007). To our knowledge, this is the first report on the presence of A. alternata affecting B. perennis plants in China. The discovery of this new disease is beneficial to the application and protection of B. perennis, which is a popular landscape and medicinal plant.

Ultrasensitive Fluorometric Angling Determination of Staphylococcus aureus in Vitro and Fluorescence Imaging in Vivo Using Carbon Dots with Full-Color Emission.

Rapid, accurate, and safe screening of foodborne pathogenic bacteria is essential to effectively control and prevent outbreaks of foodborne illness. Fluorescent sensors constructed from carbon dots (CDs) and nanomaterial-based quenchers have provided an innovative method for screening of pathogenic bacteria. Herein, an ultrasensitive magnetic fluorescence aptasensor was designed for separation and detection of Staphylococcus aureus (S. aureus). Multicolor fluorescent CDs with a long fluorescent lifetime (6.73 ns) and high fluorescence stability were synthesized using a facile hydrothermal approach and modified cDNA as a highly sensitive fluorescent probe. CD fluorescence was quenched by Fe3O4 + aptamer via fluorescence resonance energy transfer (FRET). Under optimal conditions, the FRET-based aptasensor can detect S. aureus accompanied by a wide linear range of 50-107 CFU·mL-1 and a detection limit of 8 CFU·mL-1. Compared with other standard methods, this method was faster and more convenient, and the entire test was finished within 30 min. The capability of the aptasensor was simultaneously investigated on food samples. Additionally, the developed CDs exhibited excellent biocompatibility and were thus applied as fluorescent probes for bioimaging both in vitro and in vivo. This new platform provided an excellent application of the CDs for detecting and bioimaging pathogenic bacteria.