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P1-111 Development of BAX® System Real-Time PCR Assay for Shigella

Monday, July 23, 2012
Exhibit Hall (Rhode Island Convention Center)
Linda Xuan Peng, DuPont Qualicon, Wilmington, DE
Dan Delduco, DuPont Qualicon, Wilmington, DE
Julie Kraynak, DuPont Qualicon, Wilmington, DE
Jackie Harris, DuPont Qualicon, Wilmington, DE
Lois Fleck, DuPont Qualicon, Wilmington, DE
Andrew Farnum, DuPont Qualicon, Wilmington, DE
Introduction: Shigella is the third most common pathogen transmitted through foods (higher than E. coli O157:H7). The infective dose of Shigella is as low as 10 cells, and there are an estimated 150 million illnesses and 600,000 deaths caused annually by this organism. In order to meet the urgent need for the detection of Shigellain foods, a Scorpion™ probe-based real-time PCR assay and an accompanying culture confirmation method were developed.

Purpose: The objectives of this study were to develop this BAX® System real-time PCR assay for detecting Shigella in a variety of food samples and evaluate Shigella selective media for confirmation, isolation and further characterization of Shigellaspecies.

Methods: After enrichment, lysis was performed according to the standard BAX® System method and a full process was run in the Q7 instrument. Inclusivity, exclusivity and sensitivity were tested in 123 Shigella strains, 71 closely related enteroinvasive E. coli (EIEC) strains and 81 non-Shigella/EIEC strains, and PCR tablet lot-to-lot comparison was also performed. Both commercial and non-commercial Shigellaculture media were evaluated with the inclusivity and exclusivity panel strains.

Results: The BAX® System assay followed by Shigella selective agar confirmation method demonstrated 100% inclusivity for the 123 Shigella strains tested and 100% exclusivity for the 152 non-Shigellastrains tested. The Real Time PCR assay was sensitive enough to detect 50 CFU/ml of the target pathogen both in pure culture and food samples. Consistent results were obtained by the lot-to-lot PCR assay comparison.

Significance: This developmental Real-Time PCR assay can detect Shigella species at levels as low as 50 CFU/ml. Assisted by a culture confirmation method, which helps distinguish Shigella from EIEC strains, this protocol provides a useful tool for food companies/industries to rapidly detect Shigella species in foods.

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