Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system

Area of Science:

• Food Microbiology
• Molecular Diagnostics
• Public Health

Background:

• Pathogenic *Escherichia coli* (E. coli) pose significant public health risks.
• Accurate detection and differentiation of E. coli pathotypes are crucial for food safety surveillance.
• Existing methods may lack the comprehensive screening capabilities required for multiple pathotypes.

Purpose of the Study:

• To develop and validate a novel combinatory SYBR Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli).
• To enable simultaneous detection and discrimination of five key E. coli pathotypes: STEC, EPEC, EAggEC, EAggSTEC, and EIEC.
• To assess the system's performance, including selectivity, sensitivity, repeatability, and reproducibility, for application in food matrices.

Main Methods:

• Design of 13 SYBR Green qPCR assays targeting specific virulence genes (uidA, ipaH, eae, aggR, aaiC, stx1, stx2).
• Utilized dual primer pairs per target to ensure detection despite gene deletions or polymorphisms.
• Evaluated primer pair selectivity, sensitivity, repeatability, and reproducibility in silico and in situ.
• Tested the CoSYPS Path E. coli system on artificially contaminated food matrices.

Main Results:

• Achieved 100% selectivity for all targeted genes.
• Demonstrated a limit of detection between 1 and 10 genomic copies for all assays.
• Confirmed high repeatability and reproducibility, meeting European standards.
• Successfully detected as low as 2-7 CFU of STEC per 25g of food matrix after 24h enrichment.

Conclusions:

• The CoSYPS Path E. coli system provides a sensitive, specific, and robust method for detecting and differentiating pathogenic E. coli in food.
• This multiplex qPCR approach streamlines the screening process, enhancing food safety monitoring capabilities.
• The system's performance indicates its potential for routine application in food testing laboratories.