Additional Og-typing PCR techniques targeting E. coli-novel and Shigella-unique O-antigen biosynthesis gene clusters.

The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods, such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types including OgN32, OgN33, and OgN34 presented in this study, we designed additional 24 PCR primer pairs targeting 14 novel and two diversified E. coli Og-types and eight Shigella-unique Og-types. Subsequently, we developed five new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and nine primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported herein can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.