Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E. coli infection in humans

Abstract

Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

Fig 1. E. coli O5 and O76 O-antigen gene clusters.

All genes are transcribed in a direction from the JUMPstart sequence to gnd.

Fig 2. Agarose gel electrophoresis of the PCR products obtained from E. coli type strains belonging to the 21 covered serogroups by using the three multiplex PCR assays.

(a) Multiplex 1: lanes 1 and 9, 100 bp DNA ladder; lane 2, O5; lane 3, O91; lane 4, O26; lane 5, O103; lane 6, O145; lane 7, O121; lane 8, O111. (b) Multiplex 2: lanes 1 and 9, 100 bp DNA ladder; lane 2, O55; lane 3, O128; lane 4, O113; lane 5, O146; lane 6, O76; lane 7, O45; lane 8, O177. (c) Multiplex 3: lanes 1 and 9, 100 bp DNA ladder; lane 2, O157; lane 3, O15; lane 4, O104; lane 5, O118; lane 6, O123; lane 7, O165; lane 8, O172.