Endonuclease restriction-mediated real-time PCR for simultaneous detection of Listeria monocytogenes and Listeria ivanovii

Abstract

Listeria monocytogenes and Listeria ivanovii have been identified as the only two pathogenic species in Listeria genus. Food contamination with L. monocytogenes is a severe public health issue causing great economic loss, while L. ivanovii contamination is uncommon, but represents a potential risk of infection in immunocompromised people. The sensitive and rapid method for detection of L. monocytogenes and L. ivanovii is beneficial to medical diagnosis and food industry. Herein, we developed an endonuclease restriction-mediated real-time PCR (ET-PCR) assay, allowing for concomitant detection of L. monocytogenes and L. ivanovii. This novel PCR assay is convenient in application due to the simplicity of primer design and multi-target detection. The special forward or reverse primer consisted of a reporter dye, oligonucleotides containing the recognition site of restriction enzyme BstUI, oligonucleotides for PCR amplification and a quenching dye. This assay achieved multi-target analysis by restriction of enzyme digestion and release of different reporter dyes. As few as 80 femtogram target DNA per reaction can be detected by both single and duplex ET-PCR assays, which were 100-fold more sensitive than normal PCR assay. This duplex ET-PCR assay was able to discriminate L. monocytogenes and L. ivanovii from other common foodborne pathogens. Combined with enrichment, this novel assay offers a sensitive and feasible method for detection of L. monocytogenes and L. ivanovii from different matrices. It may be an efficient tool for epidemiologist and clinician in their effort to prevent and control the infections caused by L. monocytogenes or L. ivanovii.