Identification of Salmonella H antigens by xTAG technology / 中华微生物学和免疫学杂志

ABSTRACT

Objective To develop a microsphere-based suspension array for simultaneous detec-tion and identification of Salmonella H antigens by using Luminex xTAG technology and to evaluate its capa-bility in serotyping Salmonella strains. Methods The fliC and fljB genes, encoding the H antigen of Salmo-nella, were selected as the target genes. Universal upstream primers were designed based on the highly con-served regions of fliC and fljB genes, and the corresponding specific reverse primers were designed based on the variable regions. While synthesizing, the 5′end of each upstream primer was labeled with biotin and the 5′end of each specific reverse primer was modified with its certain TAG sequence. After amplified and la-beled with biotin and TAG sequence, the PCR products of specimens were hybridized with the mixture of va-rious MagPlex-xTAGTM microspheres. Each set of microspheres contained its unique anti-TAG sequences. The results of hybridization were analyzed by using Luminex MagPix reader system and the median fluores-cence intensity ( MFI) was reported. The H antigens of 145 Salmonella strains were identified with this de-veloped xTAG suspension array, and the results were compared with those obtained by using traditional ser-um agglutination test. Results The PCR products of different H antigens ranged from 94 bp to 245 bp and could be identified by hybridizing with MagPlex-xTAGTM microspheres. There was no cross-reaction between different H antigens or with DNAs derived from Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus and Shigella flexneri. Compared with the traditional serum agglutination test, the sensitivity and specificity of the xTAG suspension array in the identification of H antigens of 145 Salmonella strains were 95. 1% and 100%, respectively. Conclusion The developed xTAG suspension array was a specific, accurate and effective method for simultaneous detection and identification of 31 H antigens of common Salmonella serovars strains. It could be used for determining the H antigens of more than 90 Salmonella strains within 5 hours.