ABSTRACT
To confirm the B cell epitope recognized by monoclonal antibody (MAb) 3G11 of bluetongue virus type 8 (BTV-8) VP2 protein prepared in our laboratory, antigen epitopes recognized by 3G11 were screened and identified by phage display technology. KLLAT sequence was found by sequencing of blue spot after four rounds panning and 283LL284 of common short peptide sequence was obtained after comparison to amino acid sequence of BTV-8 VP2 protein. The peptide sequences KLLAA, KALAT, KLAAT and KLLAT were synthesized and identified by indirect ELISA. KLLAA and KLLAT bound strongly with supernatant and as cites of 3G11 cells and reacted specifically with BTV-8 positive standard sera. Further sequence analysis showed that amino acid sequence 283LL284 was conserved among different serotypes of BTV-8 strains, and283LL284 was the key amino acids of antigen epitopes recognized by 3G11. This study laid the foundation to establish type 8 BTV specific immunological detection methods.
ABSTRACT
To develop a clinical diagnosis technique for bluetongue virus infection, we established serotype-specific methods to detect serotype 4 of bluetongue virus (BTV-4). Two monoclonal antibodies (mAbs) against VP2 protein of BTV-4, named 4A-1G7 and 4B-1B6, were used as competitive antibodies in the competitive enzyme-linked immunosorbent assays (C-ELISA). We detected 50 negative serum samples from sheep, goats and cattle by C-ELISA. The cut-off values of 4A-1G7 and 4B-1B6 mAbs were 49% and 40%, respectively. The results of the sensitivity, specificity and repeatability by detecting standard positive serum, were consistent with the general standard of Office International Des Epizooties. Furthermore, serum samples of BTV-4, BTV-18 and BTV-20 infection could be screened out through the combined C-ELISAs by 4A-1G7 and 4B-1B6 mAbs. Thus, this technique may diagnose BTV-4, BTV-18 and BTV-20 infections.
ABSTRACT
Vibrio cholerae is an important foodborne pathogen, mainly causes acute intestinal infectious disease. The development of rapid method for detecting Vibrio cholerae is critical for early diagnosis of its infection. In this study, two pairs of specific primers were designed according to housekeeping gene mdh of Vibrio cholerae. Following optimization of the reaction, DNA loop-mediated isothermal amplification (LAMP) for rapidly detecting Vibrio cholerae was successfully established. The optimal reaction for the LAMP assay is 65 degrees C for 60 min, with detection limit for cultivated Vibrio cholerae of 25 CFU/mL and for its contaminated food of 32 CFU/g. The specificity of the assay was determined using thirty-three kinds of same species or closely related bacteria, only Vibrio cholerae strains were specifically amplified. In practice, 85 pieces of positive samples were detected from 1057 pieces of shrimps, crabs, oysters, meat and human diarrhea complex using the LAMP method, which accorded with the detection result by ISO TS 21872-1-2007. Thus, the LAMP assay established in this study is a sensitive, rapid and simple tool for detecting Vibrio cholerae and will facilitate the surveillance for its control.