Abstract
Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.
Highlights
Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of diseases causing infections, including urinary tract infections, diarrheal disease, and other clinical symptoms
Four of them are associated with food, i.e. enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), and enteroinvasive Escherichia coli (EIEC)
It is important that multiplex rt-PCR conditions using SYBR Green as a label do not generate nonspecific products that give an unexpected peak as background in the melting curve result
Summary
Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of diseases causing infections, including urinary tract infections, diarrheal disease, and other clinical symptoms Six types of this bacteria are known as diarrheagenic E. coli (DEC). Multiplex real-time PCR (m-rtPCR) could separates and quantifies E. coli strains based on virulence genes. By this method, two or more sequence targets are amplified by using several pairs of primers in the same reaction. The multiplex method provides more advantages compared to simplex identification It is more efficient and faster in time detection. This study aims to evaluate a fluorescence-based (SYBR Green I) multiplex rt-PCR for detecting four types of diarrheagenic E. coli, i.e. ETEC, EPEC, EHEC, and EIEC based on their virulence factors
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