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

Abstract

Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri.

Fig. 1.

Chemical composition and specific O-antigen modification genes of different serotypes of S. flexneri. The basic O antigen consists of repeating units of tetrasaccharide. Serotypes differ by the addition of either glucosyl or O-acetyl groups to different sugars within the tetrasaccharide repeat unit via the linkages indicated. Specific O-antigen modification genes for different serotypes are indicated in parentheses.

Fig. 2.

Multiplex PCR products of S. flexneri reference strains representing all 15 serotypes. PCR products were electrophoresed on a 1.5% (wt/vol) agarose gel, stained with ethidium bromide, and photographed under UV light. Serotypes are indicated above the lanes. M1 and M2, 150- and 100-bp DNA ladder markers (TaKaRa, Japan).