Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein

Abstract

Background Salmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit. Results The recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 μg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 μg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%. Conclusion Anti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Comparative Proteomic Analysis of Secretory Proteins of Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides Investigates Virulence and Discovers Important Diagnostic Biomarkers

“…Then, 1 μg of each purified recombinant protein (Ts1133, Ts0029, Ts0707, Ts0085, and Ts0484) was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Immun-Blot ® , USA). After blocking, recombinant proteins were probed with five serum samples of calves naturally infected with Mmm, kindly provided by Prof. Jiuqing Xin from China National CBPP Reference Laboratory, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences [ 25 ], whereas sera from non-infected calves were used as the negative control. Then, the Western blotting assay was developed with HRP-conjugated goat anti-bovine IgG (Southern Biotech Co., USA) for 1 h and finally visualized with WesternBright™ ECL (Advansta, CA, USA) [ 26 ].…”

“…In brief, 96-well microtitre plates were coated overnight at 4 °C with 200 ng of each purified recombinant protein diluted in 100 μL sodium carbonate buffer (pH 9.6) and washed with PBS containing 0.05% Tween 20 (PBST). After blocking, the plates were probed for 1 h at 37 °C with sera collected from M. bovis and Mmm naturally infected calves [ 25 , 27 ]. After washing with PBST, the plates were incubated for 1 h at 37 °C with goat anti-bovine IgG-HRP (1:5000) (Southern Biotech Co., USA) and washed with PBST, followed by the addition of tetramethylbenzidine (TMB)/H 2 O 2 (Wuhan Keqian Biological Co., Ltd., China) as a substrate.…”

Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella

“…Yet, they are time-consuming and laborious, taking at least 2 days to obtain the final results . Alternatively, nucleic acid amplification techniques (e.g., polymerization chain reaction, PCR) and immunologically based methods (e.g., enzyme-linked immunosorbent assay, ELISA) are the common methods for rapid screening bacteria in the laboratory. Even sensitive enough, the nucleic acid amplification techniques require professional operations and expensive instruments .…”