Vibrio parahaemolyticus is as an important food-borne pathogen circulating in China. Since 1996, the core serotype has become O3:K6, which has specific genetic markers. This serotype causes the majority of outbreaks worldwide. Until now, nearly 21 serotypes were considered as serovariants of O3:K6. Among these, O4:K68, O1:K25 and O1:KUT have caused pandemic outbreaks. O4:K8, a serovariant of O3:K6, has become the second most dominant serotype circulating in China after O3:K6. In this study, we report the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 146 V. parahaemolyticus isolates belonging to 23 serotypes. Upon mass spectral analysis, isolates belonging to O4:K8 formed a distinct group among the five main pandemic groups (O3:K6, O4:K8, O4:K68, O1:K25 and O1:KUT). Two major protein peaks (m/z 4383 and 4397) were significantly different between serotype O4:K8 and the four other pandemic strains. Both of these peaks were present in 32 out of 36 O4:K8 isolates, but were absent in 105 out of 110 non-O4:K8 isolates. These peaks were also absent in all 74 pandemic serotypes (O3:K6, O4:K68, O1:K25 and O1:KUT). Our results highlight the threat of O4:K8 forming a distinct group, which differs significantly from pandemic serotypes on the proteomic level. The use of MALDI-TOF MS has not been reported before in a study of this nature. Mass spectrum peaks at m/z 4383 and 4397 may be specific for O4:K8. However, we cannot conclude that MALDI-TOF MS can be used to serotype V. parahaemolyticus.

Ricin and abrin are phytotoxins that can be easily used as biowarfare and bioterrorism agents. Therefore, developing a rapid detection method for both toxins is of great significance in the field of biosecurity. In this study, a novel nanoforest silicon microstructure was prepared by the micro-electro-mechanical systems (MEMS) technique; particularly, a novel microfluidic sensor chip with a capillary self-driven function and large surface area was designed. Through binding with the double antibodies sandwich immunoassay, the proposed sensor chip is confirmed to be a candidate for sensing the aforementioned toxins. Compared with conventional immunochromatographic test strips, the proposed sensor demonstrates significantly enhanced sensitivity (≤10 pg/mL for both toxins) and high specificity against the interference derived from juice or milk, while maintaining good linearity in the range of 10–6250 pg/mL. Owing to the silicon nanoforest microstructure and improved homogeneity of the color signal, short detection time (within 15 min) is evidenced for the sensor chip, which would be helpful for the rapid tracking of ricin and abrin for the field of biosecurity.

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

Ricin (RT) and abrin (AT) are plant toxins extracted from Ricinus communis and Abrus precatorius, respectively, and both have N-glycosidase activity. The detection of these toxins is vital because of their accessibility and bioterrorism potential. While ricin can be effectively detected based on its depurination activity, only a few tests are available for detecting the depurination activity of abrin. Therefore, it is unclear whether they share the same optimal reaction substrate and conditions. Here, we established optimum depurination conditions for ricin and abrin, facilitating the in vitro detection of their depurination activity using high-performance liquid chromatography–tandem mass spectrometry. The parameters optimized were the reaction substrate, bovine serum albumin (BSA), buffer, pH, temperature, time, antibodies, and magnetic beads. Both toxins showed better depurination with single-stranded DNA. However, substrate length, adenine content, BSA concentration, buffer concentration, reaction temperature, and reaction time differed between the two toxins. The optimal conditions for ricin depurination involved a reaction in 1 mM ammonium acetate solution (0.5 μM DNA15A, 20 μg/mL BSA, and 1 mM Zn²⁺, with pH 4.0) at 55 °C for 1 h. The optimal conditions for abrin depurination involved a reaction in 1 mM ammonium citrate solution (0.2 μM DNA20A, 10 μg/mL BSA, 1 mM Mg²⁺, and 0.5 mM EDTA, with pH 4.0) at 45 °C for 2 h. After optimization, the limits of detection (LOD) for ricin and abrin were 0.506 ng/mL and 0.168 ng/mL, respectively. The detection time was also significantly reduced.

Epsilon toxin (ETX), produced by type B and D strains of Clostridium perfringens, can cause fatal enterotoxaemia in ruminant animals, particularly sheep, cattle, and goats. Previous studies show that the cytotoxicity of ETX is dependent on the integrity of lipid rafts, the maintenance of which is ensured by cholesterol. Zaragozic acid (ZA) is a statin drug that reduces the synthesis of squalene, which is responsible for cholesterol synthesis. In this study, ZA significantly reduced the toxicity of ETX in Madin–Darby canine kidney (MDCK) cells. We show that ZA does not affect the binding of ETX to MDCK cells, but propidium iodide staining (PI) and Western blotting confirmed that ZA significantly disrupts the ability of ETX to form pores or oligomers in MDCK cells. Additionally, ZA decreased the phosphatidylserine exposure on the plasma membrane and increased the Ca²⁺ influx of the cells. Results of density gradient centrifugation suggest that ZA decreased the number of lipid rafts in MDCK membranes, which probably contributed to the attenuation of pore-formation. Moreover, ZA protected mice against ETX in vivo. All mice pre-treated with ZA for 48 h before exposure to an absolute lethal dose of ETX (6400 ng/kg) survived. In summary, these findings provide an innovative method to prevent ETX intoxication. Considering many pore-forming toxins require lipid rafts, we tested and found ZA also inhibited the toxicity of other toxins such as Clostridium perfringens Net B and β-toxin (CPB) and Staphylococcus aureus α-hemolysin (Hla). We expect ZA can thus be developed as a broad-spectrum medicine for the treatment of multiple toxins. In addition, other statins, such as lovastatin (LO), also reduced the toxicity of ETX. These findings indicate that statin medicines are potential candidates for preventing and treating multiple toxin-induced diseases.

Food poisoning caused by Staphylococcal enterotoxins (SEs) is prevalent globally, making efficient detection of these toxins very important. Traditionally, liquid chromatography–mass spectrometry required immunosorbent enrichment by magnetic bead-coupled antibodies obtained by animal-specific immunization. However, this method is time-consuming and costly. In this study, two recombinant protein capture molecules were designed based on the principle of toxins binding to Major Histocompatibility Complex (MHCII) and T cell receptor (TCR) molecules. The two capture molecules are called MHCII and MHCII-D10. The design of the MHCII and TCR-D10 was achieved through searching for the binding site protein sequence of Staphylococcal enterotoxins in the relevant literature, and MHCII-D10 was to link MHCII sequence with TCR-D10 sequence using linker (G4S)3 linking peptide. These capture molecules were shown to effectively bind to seven types of toxins and to capture SEs in various matrices. The digestion time, ratio, and temperature were further optimized, reducing the overall digestion time to just 2 h. The specificity, linearity, sensitivity, precision (RSD%), and recovery of the two methods were verified by liquid chromatography–mass spectrometry. When the MHCII and MHCII-D10 captured the toxins, the limit of quantification (LOD) in the 1 × PBS, plasma, and milk matrices ranged from 1.5625 to 100 fmol/µL, with the recovery rate ranging from 18.4% to 96%. The design of these capture molecules eliminates the need for animal-specific immunization, simplifying the pre-detection process and avoiding ethical concerns. This development holds significant promise for clinical diagnosis and reference.