Innovative Automated Sample Prep to Reduce the Positive Rate of Non-O157 STEC

Introduction: Detection of non-O157 STEC is based on PCR detection of virulent associated genes (stx and eae), Top6 O-groups. The prevalence of stx, eae and O-groups associated genes in food samples leads to positive results due to the detection of genes carried by different strains. The association of the VIDAS (ESPT, Phage Technology) for the capture of O-groups of interest upstream the PCR allows a better specificity for the detection of non-O157 STEC.

Purpose: Compare the VIDAS ESPT+PCR method to commercial PCR for the detection of non-O157 STEC in food samples

Methods: 376 natural raw milk products were analyzed. This food category is known to contain various Escherichia coli strains harboring stx or eae genes only, leading to high positive rate with PCR methods. Twenty-five g were diluted 1/10 in BPW+Acriflavine (10mg/l), homogenized, and incubated 18h at 37°C. DNA was extracted using VIDAS ESPT1 protocol or commercial lysis kit from 800µl and 50µl of enrichment broth, respectively. DNA was analyzed by PCR screen 1 (stx and eae), and tested for O-groups (Top6) when positive for both stx and eae.

Results: 4/376 samples were tested positive for both stx and eae when DNA was prepared using VIDAS ESPT1. Those 4 samples were shown positive as well for O-groups. The commercial PCR method on crude extract shown 37 positive samples for both stx and eae; 21/37 were positive for O-groups PCR. Among those 376 samples, an O26 strain (eae and stx-positive) was detected by both methods and successfully isolated.

Significance: The VIDAS ESPT automated DNA sample preparation prior to PCR enables a reduction of about 89% of positive rate for non-O157 STEC detection, limiting the number of samples to be analyzed for O-Groups.