Polymerase chain reaction detection of haemolysin D gene (hlyD) in uropathogenic Escherichia coli as a novel diagnostic test for urinary tract infection

Abstract

Background: Urinary tract infection (UTI) is a common and sometime serious infectious disease diagnosed using conventional urine culture as the ‘gold standard’ for identifying Escherichia coli, the most common causative agent. However, due to the slow turn-around-time and other challenges of urine culture, this study explores the use of a novel biomolecular polymerase chain reaction (PCR) approach to detect the presence of haemolysin D gene (hlyD) that encodes a unique virulence factor of uropathogenic E. coli (UPEC) for its rapid identification in UTI.

Methodology: Primers from UPEC CFT073 and non-pathogenic E. coli K-12 MG1655 strains provided by Nottingham Trent University, England, UK were used to investigate the presence of haemolysin D gene (hlyD) in UPEC. The hlyD primers were developed from hlyD with locus number C_RS01660 on UPEC CFT073 strain using the NCBI, virulence finder, and Island viewer, and used in a PCR assay to target the hlyD in UPEC. Three sets of PCR templates were designed (UPEC, E. coli, and “No template”), each with internal and external controls amplified in a multiplex PCR assay, and agarose gel electrophoresis was used to separate the amplicons, and determine the specificity of hlyD for UPEC.

Results: The UPEC genome PCR assays were positive for hlyD and UPEC positive control, and similarly, PCR was positive for E. coli genome positive control, but negative for hlyD. Moreover, the “No template” PCR assay was clean with no amplification product, confirming the absence of PCR contaminations.

Conclusion: The hlyD is a unique virulence gene specific for UPEC. PCR assay of this gene is a promising specific and rapid biomolecular diagnostic test that can overcome the limitations of the traditional approaches for detection of UPEC in UTI.