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.
Highlights
Shiga toxin (Stx)-producing Escherichia coli (STEC) are important food-borne zoonotic pathogens responsible for a broad spectrum of clinical symptoms in humans, ranging from mild diarrhea to hemorrhagic colitis (HC) and the life-threatening hemolytic uremic syndrome (HUS) [1]
Serotype O157:H7 has been implicated in most outbreaks and in most cases of HUS, there is growing concern about the risk to human health associated with nonO157 Shiga toxin-producing E. coli (STEC) serotypes [2,3], which may be as well responsible for important outbreaks, such as the renowned 2011 STEC O104:H4 German outbreak [4]
The majority of clinical STEC infections, those associated with outbreaks and serious patient outcomes, are attributable to strains belonging to a subset of STEC serotypes called enterohemorrhagic E. coli (EHEC)
Summary
Shiga toxin (Stx)-producing Escherichia coli (STEC) are important food-borne zoonotic pathogens responsible for a broad spectrum of clinical symptoms in humans, ranging from mild diarrhea to hemorrhagic colitis (HC) and the life-threatening hemolytic uremic syndrome (HUS) [1]. 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 suitable for being used in PCR assays for serogroup identification.
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