Rapid Species-Specific Identification of Listeria Isolates Using Multiplex PCR

Introduction: Standard commercially available test methods used by the food industry target either the genus Listeria or Listeria monocytogenes, directly. These results must be confirmed using culture methods. However, these confirmation methods are time consuming, require multiple steps, and cannot simultaneously differentiate between multiple species of Listeria.

Purpose: This study was performed to develop a novel multiplex PCR method that will provide rapid, identification of Listeria spp. within a single test.

Methods: Six species-specific primer sets targeting hly, iap, lse24, smcL, Lwe1801, and one genus-specific primer set targeting 23S rRNA were used for simultaneous differentiation of L. monocytogenes, L. innocua, L. grayi subsp. grayi, L. grayi subsp. murrayi, L. seeligeri, L. ivanovii, L. welshimeri, and the genus Listeria, respectively. DNA extracted from 13 Listeria spp. ATCC strains and 64 unknown presumptive Listeria spp. isolates were tested by the multiplex PCR method to evaluate the efficiency of the selected primer sets. Species identification was determined by gel electrophoresis based on the designated size (bp) of the species-specific PCR product.

Results: The multiplex PCR method simultaneously amplified both a species and a genus specific PCR product per strain and successfully identified all 13 ATCC strains of Listeria. Additionally, this method identified 22 unknown isolates as L. monocytogenes (hly ~713 bp), 42 isolates as L. innocua (iap ~975-987 bp), and as expected demonstrated that all 64 isolates were within the genus Listeria (23S rRNA ~77bp). Overall, the multiplex PCR method was efficient and 100% accurate for species-specific identification of Listeria.

Significance: This new multiplex PCR method offers rapid, identification of presumptive Listeria spp. isolates. This knowledge will enhance risk assessments and provide information regarding the prevalence and potential co-existence of each species of Listeria in food products and the food production environment.