Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probe-based real-time PCRs

Abstract

Introduction: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood. Material and Methods: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted. Results: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products. Conclusion: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.