P1-46 Enrichment Temperature Variation Effects on Shigella qPCR in High Background Food Matrices

Monday, July 29, 2013
Exhibit Hall (Charlotte Convention Center)
Amie Minor, West Virginia Department of Agriculture, Charleston, WV
Kellie Littlefield, West Virginia Department of Agriculture, Charleston, WV
Brenda Keavey, West Virginia Department of Agriculture, Charleston, WV
Introduction: Shigella is the third most common foodborne related pathogen that causes illness. Current approved methods incorporate varying enrichment temperatures for Shigella detection. An effective enrichment temperature for high background matrices is necessary to produce reliable and repeatable results in low level Shigella detection using qPCR.   

Purpose: This study compares 35°C to 42°C enrichment temperatures for its effect on Shigella and high background competitive growth used in conjunction with a qPCR endpoint for detection.

Methods: To determine the effect of temperature on the flora of high background matrices as well as the enumeration of Shigella species in Shigella broth, raw ground beef samples were enriched with Shigella broth.  Duplicates were run in parallel at 35°C and 42°C overnight.  Enrichment broths (non-matrix) were also fortified with a fresh inoculum of approximately 20 CFU of Shigella flexneri 2457M. 

Two sample sets were analyzed on consecutive days with raw ground beef samples that contained varying fat contents and bacterial loads.  Samples were fortified at levels of 2.0 – 0.2 CFU/g at intervals of 0.2.  Parallel samples were enriched at 35°C and 42°C incubations.  Samples were analyzed following the FERN-MIC.0013.01 qPCR method in duplicate using the Smart Cycler II with ipaH and IC gene targets.

Results: Overall the mean ipaH target threshold values for 42°C were 5.93 (27.01vs.32.94) cycles shorter than those at 35°C and were consistent throughout each inoculum level.  Data generated indicated that Shigella spp. tend to grow slower in enrichment at 42°C vs. 35°C by nearly a half log (1.0x109 vs. 1.5x109) but the 42°C enrichment reduced the amount of background competitors by an entire log (4.0x108 vs. 1.6x109 CFU/g). 

Significance: The results presented here indicate a 42°C enrichment temperature for Shigella broth is more effective than 35°C for inhibiting the growth of background competitors in high background matrices and thus demonstrated a lower limit of detection for endpoint qPCR.