T4-10 Culture-independent Detection and Confirmation of Shiga Toxin-producing Escherichia coli by Digital PCR

Monday, July 10, 2017: 4:15 PM
Room 16 (Tampa Convention Center)
Jianfa Bai , Kansas State University , Manhattan , KS
Xuming Liu , Kansas State University , Manhattan , KS
Lance Noll , Kansas State University , Manhattan , KS
Xiaorong Shi , Kansas State University , Manhattan , KS
T G Nagaraja , Kansas State University , Manhattan , KS
Gary Anderson , Kansas State University , Manhattan , KS
Introduction:  Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens. None of the current PCR assays can confirm whether a detected virulence gene is associated with a particular serogroup of STECs from complex matrices. The current USDA-FSIS recommended method takes one week to result due to confirmation through cultural isolation; this does not fit high throughput settings.

Purpose: This study was undertaken to develop a digital PCR assay for the detection and confirmation of major STECs without cultural isolation.

Methods: Seven major STEC serogroups, O26, O45, O103, O111, O121, O145, and O157, were used as pure cultures and to inoculate cattle feces with three replications. For each serogroup, a strain that carried both the serogroup-encoding gene and an stx gene was compared to a mixture of two strains (each carried one of the two genes) using Thermo Fisher QuantStudio 3D and Fluidigm Biomark digital PCR systems. The assay was also evaluated with 100 cattle fecal samples.

Results: The gene association rates (GAR, percentage of dual signals) by QuantStudio 3D for the O157 strain that carried O157-antigen gene and stx2 was 62.5% for culture and 77.2% for spiked feces; GAR for the mixture of two strains was 21.2% for culture and 28.3% for spiked feces. GARs by Fluidigm Biomark for each of the seven major STECs that carried two genes ranged 67.3 to 90.5% for culture and 74.6 to 88.0% for culture-spiked fecal samples. GARs for the two genes carried by separate genomes were between 8.7 and 28.8% for culture, and 8.3 to 30.9% for culture-spiked fecal samples. A strain carrying three genes (rfbE-O157, stx2, and eae) was correctly identified. Three O103 strains that carried eae and three O45 strains that carried stx1 genes were identified from a collection of 100 cattle fecal samples.

Significance: Our digital PCR assay is a culture-independent and high throughput assay. It is capable of detecting STECs in only two days.