The detection of the genus Listeria pathogenic bacteria by real-time polymerase chain reaction

Listeriosis is an infectious disease in humans and animals caused by bacteria of the genus Listeria. Currently, different PCR test systems manufacturers offer reagent kits for the qualitative and quantitative detection methods exclusively of L. monocytogenes in biological material. In the study a universal protocol for the identification of bacterial strains, etiological agents of listeriosis - L. monocytogenes, and L. ivanovii is described. The method allows finding microorganisms of the genus Listeria using real-time polymerase chain reaction (Real-time PCR) based on the genes fragments amplification encoding the pathogenicity factors of these species bacteria. Screening for collection strains selected unique fragments of the inlA and smcL target genes encoding L. monocytogenes internalin A and L. ivanovii sphingomyelinase C, respectively. The optimal conditions for the listeriosis pathogen detection using Real-time PCR were established based on the primers selected nucleotide sequences. The sensitivity of the genus Listeria pathogenic bacteria Real-time PCR was established. For L. monocytogenes, it made 3 × 102, and for L. ivanovii 3 × 103 bacterial cells in 1 ml. The specificity of Real-time PCR for L. ivanovii and L. monocytogenes strains detection was defined on the basis of genes of smcL and inlA. For this purpose besides the strains of L. ivanovii and L. monocytogenes the strains of several types of Listeriya (L. innocua, L. seeligeri, L. welshimeri), and also isolates of other genus of bacteria were used. The described protocol is genus specific for the identification of the genus Listeria strains, including monocytogenes, and also allows finding L. ivanoviiс with the help of genus specific target directly.