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Food Testing >> Resources >> An Automated Highly Multiplexed PCR Method for Listeria Fingerprinting Also Provides Improved Confirmation Rates with Presumptive Environmental Sample Enrichments

An Automated Highly Multiplexed PCR Method for Listeria Fingerprinting Also Provides Improved Confirmation Rates with Presumptive Environmental Sample Enrichments

Daniel R. DeMarco ¹, Andrew Stock ², Megan Murn ², Patricia Quinn ², Gwendolyn Spizz ³, I. Cristina McGuire ³, Brooke Schwartz ³
Eurofins Microbiology Laboratories, Inc.: ¹ Louisville, KY; ² Mounds View, MN; Rheonix, Inc. ³ Ithaca, NY

ABSTRACT

Introduction

Food producers wish to characterize Listeria spp. positive environmental samples for source tracking or trending purposes but manufacturer support for the most common platforms has declined or ended. Whole genome sequencing could provide this characterization, but US users have concerns about security and privacy of sequence files. Additionally, cultural confirmation of Listeria presumptives, continues to be a challenge. The Rheonix Listeria PatternAlert^TM method can provide both characterization and confirmation directly from presumptive sample enrichments.

Purpose

To evaluate an automated highly multiplexed PCR system for the characterization and molecular confirmation of presumptive environmental sample enrichments in comparison to cultural confirmation.

Methods

Presumptive environmental sample enrichments from a real time PCR screening method (Eurofins Gold Standard Diagnostics’ BACGene™ Listeria spp.) were tested by automated highly multiplexed PCR (1.25 mL) and with a Listeria spp. FDA-BAM-cultural method. All (excluding Facility C) samples were pre-treated with DNAse to destroy exogeneous DNA prior to real-time PCR.

Results

Of the 68 presumptives tested (note: Facility C data was not available at time of abstract submission), only 19 (28 %) were confirmed as Listeria by culture. In contrast, the highly multiplexed PCR system detected these same 19 samples plus 10 more (43%). Two unique fingerprint patterns were observed. Interestingly 7/10 presumptives that were detected by automated multiplexed PCR but not by culture were Cq < 30, suggesting Listeria was present at high levels in these samples.

Significance

The automated highly multiplexed PCR method showed superior performance to culture: detecting more presumptives, thereby reducing the non-confirming presumptive rate.