Development of a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri

Abstract

To develop a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella (S.) flexneri. Eight pairs of primer for O-antigen synthesis and modification genes of S. flexneri were designed and used for developing an O-antigen modification gene-specific singleplex PCR assay to serotype 14 most common S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Y, X, Xv and F6). Bacterial pathogens which causing diarrheal disease were used for specificity detection. 106 S. flexneri clinical isolates were serotyped by this method and compared with the slide agglutination method. An O-antigen modification, gene-specific singleplex PCR was developed. When six singleplex PCR reactions were performed, 14 of the 15 recognized S. flexneri serotypes were identified, except for serotype Xv. The detection threshold ranged from 10 pg to 1 ng DNA in a 20 µl reaction system. A high concordance between the singleplex PCR assay and slide agglutination were observed when 106 S. flexneri strains of various serotypes were analyzed with an exception that 1 serotype Y strain showed that it was carrying the additional defective gtr II genes. This method showed advantages over the traditional slide agglutination methods, and was promising when under application in the following situations as clinical diagnosis.