SYBR green real time PCR assay for quantitative detection of Escherichia coli directly from clinical samples

Abstract EN

Diagnosis of bloodstream bacterial infection in its early stages are key steps for its treatment, however because of the brutal effects of sepsis and the necessity of its immediate treatment, many physicians don’t wait for the conventional microbiological diagnosis and start prescribing antimicrobial drugs.

Therefore designing a fast, accurate and economic quantitative real-time PCR method (qR-PCR) to quantify the total number of Escherichia coli and to detect pathogenic strains of the bacterium from blood and clinical samples is a crucial step for early diagnosis and treatment of the problem.

In this study, SYBR Green I used as inexpensive indicator to reduce the costs in comparison with probe based methods and is compatible with conventional microbiological methods.

UspA gene which encodes for the universal regulator stress protein has been selected as a target gene which is the most suitable candidate to detect the presence of any E.

coli, while eae gene which encodes for the outer membrane protein intimin was targeted in order to evaluate the presence of virulence strains among positive samples.

The suitability and utility of the quantitative SGR-PCR assay for detection of E.

coli in different samples sources has been evaluated by artificial inoculation of healthy blood with type strains of E.

coli used for designing a quantitative standard curve.

The protocol was then applied on blood and some clinical samples collected from suspected patients with sepsis.

Using this method and despite of a relatively low positive data when testing the blood samples, both sets of primers showed to be useful for the detection of E.

coli and the strains that harbor virulence genes.

The developed standard curves confirmed that this method is a quantitative in a detection range of 102 to 108 cells mL-1, and that the qSG R-PCR assay can be used as a new and successful molecular tool to detect and quantify all and pathogenic E.

coli directly from clinical samples without pre-enrichment steps, when presenting at concentrations greater than 102 cells mL-1.