Development of the means for identification of dna of listeria monocytogenes types by the real-time polymarization chain reaction
DOI:
https://doi.org/10.31548/dopovidi2018.03.025Abstract
Listeriosis is a zooanthroponosis infectious disease characterized by expression of various clinical symptoms both in animals and in humans and also high mortality rates.
The peculiarity of the listeriosis is prolonged bacteriocarrier. Carriers of the pathogen may play a role in the emergence and preservation of stationary focuses of the disease. In recent years, there are evident of changes in biological properties of pathogens of food-borne toxic infections, under the influence of anthropogenic pressure. The genus of Listeria belongs to the Listeriaceae family, the classification of which is based on a phylogenetic analysis (for the indicated family in accordance with the presence of 16S rRNA). The 16 species have been included in the genus by November 2015. The L. Monocytogenes is the most relevant in the etiology of listeriosis. Its specific pathogenicity factor is listeriolysin O - hemolysin, the "main factor" of the pathogenicity of the listeria, which has a pronounced toxic effect. However, it is known that under certain conditions the activity of production of listeriolysin O decreases, which leads to the complication of microbiological diagnosis of listeriosis. The ability to produce hemolysin also has L. ivanovii and L. seeligeri. That is important to consider when conducting microbiological studies and constructing species-specific primers. The antigenic structure of the listeria consists of somatic O-antigen, flagella H-antigen. The most studied in L. monocytogenes, of which three - 4b, 1 / 2b, 1 / 2a - cause 90% of all human listeriosis, the most pathogenic is 4v. Nowadays, the polymerase chain reaction (PCR) is the one of the advanced methods for indicating pathogens of infectious diseases. That makes it possible to detect DNA of the pathogen in cases where microbiological methods do not provide identification of the pathogen identification.
The aim of the study is determination of the specificity, sensitivity and within-assay reproducibility of indication Listeria monocytogenes by real-time polymerase chain reaction.
Methods. The region of HlyA, which encodes listeriolysin, was used to design the DNA primers. The nucleotide sequence of the DNA primers and the same of the fluorescent probe The specificity of the DNA primers was determined using reference samples and field isolates of protoplast and spheroplast S-and L-form L. monocytogenes as well as strains of other genera as negative controls of Listeria Different concentrations of L. monocytogenes cultures were obtained to determine the sensitivity by serial dilutions 1 x 109 to 0.1 x 101 CFU/cm3.
The 1 cm3 of the enrichment bacteria culture had been centrifuged at 13.5 thous. rpm during 2 min and the supernatant was removed to extract nucleic acid. The bacterial residue had been resuspended in 200 µl of ТЕ buffer and then incubated in a thermostat at 95 °C during 5 min. Then the cell debris had been pelleted at 5.0 thous. rpm during 2 min and 180-190 µl of the supernatant was used for the PCR.
The amplification took place in 25 µl of reaction mixture, which included: a PCR buffer, 2.5 µmol of MgCl2, 2.0 µmol of each of the deoxynucleoside triphosphates, 0.5 µmol of forward and reverse DNA primers, 0.25 µmol of fluorescent probe (marked by FAM) and 0.25 units of Taq DNA-polymerase. The amplification was carried out with the following temperature profile: 5 min. at 94 °C to activate the polymerase and 45 cycles (0.20 sec. at 95 °C - DNA denaturation, 0.20 sec. at 56 °C – annealing the DNA primers, 0.30 sec. 72 °C – DNA chain elongation).
Results. The 9 strains were selected determine the specificity of primers to L. monocytogenes as the homologous specimens L. monocytogenes, allocated on the territory of Ukraine, Poland. The strain L. monocitogenes АТСС 19112 was used as the reference strain. Strains have 4 serovarians: 4b, 1/2a, 1/2b, 3A.
The specificity and sensitivity of the medium in relation to the L-shapes of the spheroplastic and protoplast types also were studied. Strains in the S-form had typical culturally-morphological, enzymatic, hemolytic properties for the species, (except for the L. monocytogenes 3A, in which hemolytic properties did not manifest). L-forms of spheroplastic and protoplast types have the corresponding culture-morphological and enzymatic features, they did not have hemolytic activity. Obtained primers were high specific to the investigated strains. The cross-reactions with other types of listeria or other pathogens were not noted.
The threshold of sensitivity of the methods is 1х101 CFU/cm3 for L. monocytogenes serotypes 1/2А, 1/2В, 3А, 4В in S-forms and L-forms of the protoplast and spheroplastic types.
Conclusions. The developed method for detection of DNA of bacteria Listeria monocytogenes in real-time by PCR provides the DNA isolation of the bacteria of the species Listeria monocytogenes. The threshold of detection the tool provides with the detection of DNA of spheroplast L-form Listeria monocytogenes in samples with a minimum concentration of the causative agent 1 x 101 CFU/cm3. The further research will be aimed at the validation of the Listeria monocytogenes detection tool.
Key words: Listeria monocytogenes, L-forms, biological properties, polymerase chain reaction in real time
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