Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Construction of a Colorimetric and Fluorescence Dual-Mode Immunoassay Detection of Alpha-Hemolysin in Milk.

Alpha-hemolysin (Hla) is a major virulence factor secreted by Staphylococcus aureus (S. aureus), which can lyse a variety of mammalian cells and help bacteria evade the host immune system or antibiotics, posing a safety hazard to human health. Therefore, it is critical to establish a quick-responsive and sensitive method for Hla detection to ensure food safety. In this work, a dual-mode immunoassay was developed with both colorimetric and fluorescent readouts for discriminative detection of Hla. The proposed sensing system consists of p-phenylenediamine (PPD) and fluorescein, where fluorescein functions as a fluorescent reporter, and PPD serves a dual function as a colorimetric reporter and fluorescence quencher. Subsequently, the reaction system of this method was optimized, and the detection limit, sensitivity, and specificity were evaluated. Under optimal conditions, the proposed method possesses excellent analytical performance in the range from 0.5 to 500 ng/mL with a limit of detection as low as 0.5 ng/mL. Noteworthy, this method was successfully employed for the detection of Hla in milk with good selectivity and high accuracy. Overall, the dual-mode immunoassay provides a superior platform for the on-site, quantitative, and accurate detection of Hla in food samples.

Prevalence, Antimicrobial Resistance, and Molecular Characteristics of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus from Retail Ice Cream in Shaanxi Province, China.

Staphylococcus aureus (S. aureus) is one of the major opportunistic foodborne pathogens as well as a source of human and animal infections. As surveillance of S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) is limited in ice cream, a total of 240 ice cream samples were collected from three cities in Shaanxi province, China, and screened for S. aureus. All isolates were characterized by antimicrobial susceptibility testing, staphylococcal protein A typing, multilocus sequence typing, enterobacterial repetitive intergenic consensus typing, virulence, and resistance genes. S. aureus was recovered from 10 (4.2%) ice cream samples (13 isolates) with average count from 10 to 100 colony-forming units per gram in all cases. Resistance to amoxicillin/clavulanic acid, penicillin, and trimethoprim/sulfamethoxazole (each 100.0%) was most frequently observed, followed by ampicillin (76.9%), erythromycin (46.2%), ceftriaxone (30.8%), and cefoxitin (15.4%). A total of five types of antimicrobial resistance genes were detected, including ß-lactam (blaZ and mecA), macrolide (ermB and ermC), tetracycline (tetK), aminoglycoside [aac(6')/aph(2') and aph(3')-III], and trimethoprim (dfrG). All of the strains harbored at least one staphylococcal enterotoxins gene. The commonly detected virulence genes were selw and hld (100.0%), followed by selx (92.3%); hla (84.6%); pvl (76.9%); seg, sem, and sen (each 38.5%); sei, seo, and hlb (each 30.8%); sea, seb, selu, and sely (each 23.1%); sed, sej, sek, sep, and seq (each 15.4%); and ser (7.7%). ST5-t002, ST7-t091, and ST5225-t4911 (each 15.4%) were the predominant clones, followed by ST5-t045/t105, ST6-t701/t15417, ST25-t078, ST188-t189, and ST398-t034 (each 7.7%). Among the 13 strains of S. aureus, 2 isolates were detected as MRSA (15.4%), and the molecular type belonged to ST5225-IVa-t4911. Using a 98.8% similarity cutoff, the 13 isolates were divided into 5 clusters (I-1 to I-5). These results demonstrated that the prevalence of S. aureus and MRSA was low in ice cream. However, these isolates exhibited a high level of potential pathogenicity, which represents a potential health hazard for consumers.