ISSN 2415-3060 (print), ISSN 2522-4972 (online)
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УЖМБС 2021, 6(2): 115–124
Hygiene and Ecology

Genotyping of Listeria by Polymerase Chain Reaction (PCR) and its Epidemiological Significance

Zyuzin V., Tuzova O., Frenkel U., Muntian L., Zyuzin D.

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis

Keywords: genotyping, listeria, polymerase chain reaction, epidemiological significance

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  1. Zyuzin VO, Frenkel YuD, Muntyan LYa, Tuzova ОВ. Diahnostyka ta profilaktyka listerioznoyi infektsiyi v suchasnykh umovakh [Diagnosis and prevention of listeriosis infection in modern conditions]. Olbia Forum - 2020: Strategies of the Black Sea Region in the Geopolitical Space, XIV International Scientific Conference. 2020 Jun 4-7, Mykolaiv. 2020; 2020: 5-7. [Ukrainian]
  2. Zyuzin VO, Polovenko LS, Belikova OI, Romanyuk AS, Zyuzin DV. Epidemiolohichna ta mikrobiolohichna kharakterystyka listerioznoyi infektsiyi [Epidemiological and microbiological characteristics of listeriosis infection]. Scientific Bulletin of the Nikolaev National University named after VO Sukhomlinsky. Biological sciences. 2017; 2(9): 12-17. [Ukrainian]
  3. Khramov MV, Kostenko YuG. Listeryoz: laboratornaya diahnostika v sovremennykh usloviyakh [Listeriosis: laboratory diagnostics in modern conditions]. Materials of the II National Congress of Bacteriologists. Bataevo, 2016. 2016; 6(3): 106-110. [Russian]
  4. Zyuzin VO, Frenkel YuD, Muntyan LYa, Tuzova ОВ, Frolov YuA. Henotypuvannya listeriy za dopomohoyu polimeraznoyi lantsyuhovoyi reaktsiyi (PLR) ta yoho epidemiolohichne znachennya [Genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance]. Mohyla Readings - 2020. Experience and trends in society in Ukraine: global, national and regional aspects. XXIII All-Ukrainian scientific-practical conference. Actual problems of medicine. Mykolaiv; 2020. 2020: 13-15. [Ukrainian]
  5. Samson A, Marchuk T, Filipenya E. Listerioz: etiologiya, diagnostika i lechenie [Listeriosis: etiology, diagnosis and treatment]. Liky Ukrainy. 2005; 2: 27-30. [Russian]
  6. Zyuzin VO, Frenkel YuD, Muntyan LYa, Tuzova OV. Epidemiolohichni osoblyvosti listeriozu yak zooantroponoznoho zakhvoryuvannya [Epidemiological features of listeriosis as a zooanthroponotic disease]. Olbia Forum - 2020: Strategies of the Black Sea Region in the Geopolitical Space, XIV International Scientific Conference. 2020 Jun 4-7, Mykolaiv. 2020; 2020: 3-5. [Ukrainian]
  7. Spanu C, Scarano Ch, Ibba M, Spanu V, Pietro E, De Santis L. Occurrence and traceability of Listeria monocytogenes stains isolated from sheep’s milk cheese-making plants environment. Departament of Veterinari Medicine, University of Sassari, Via Vienna 2, 07100 Sassari, Italy. Food control. 2015; 47: 318-325.
  8. Ministry of Health of the USSR. Woof. Sanitary-epidemiological management. Metodicheskie ukazaniya po sanitarno-bakteriologicheskomu kontrolyu na predpriyatiyakh obshchestvennogo pitaniya i torgovli pishchevymi produktami [Methodical instructions for sanitary and bacteriological control at public catering and food trade enterprises]. M; 1986. 61 p. [Russian]
  9. Ministry of Health of the USSR, Order No. 535, 1985 Apr 22. Ob unifikatsii mikrobiologicheskikh (bakteriologicheskikh) metodov issledovaniya, primenyaemykh v kliniko-diagnosticheskikh laboratoriyakh lechebno-profilakticheskikh uchrezhdeniy [On the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions]. M; 1985. 123 p. [Russian]
  10. USSR, Ministry of Health from 04.09.1986. Gosagroprom of the USSR from 02.13.1987. Metodicheskie rekomendatsii po laboratornoy diagnostike listerioza u zhivotnykh i lyudey [Guidelines for laboratory diagnosis of listeriosis in animals and humans]. M; 1987. 67 s. [Russian]
  11. Ministry of Health of Ukraine. Metodicheskie rekomendatsii. O poryadke provedeniya laboratornykh issledovaniy na listerioz v uchrezhdeniyakh sanitarno-epidemiologicheskoy sluzhby pri pishchevykh toksikoinfektsiyakh [Guidelines. On the procedure for conducting laboratory tests for listeriosis in the institutions of the sanitary and epidemiological service for food toxicoinfections]. Kharkov; 1995. 75 s. [Russian]
  12. Isolation and identification of Listeria monocytogenes. Ed by GL Jones. US Department of health and human services, CDC. Atlanta-Georgia; 1989. 53 p.
  13. Shapoval VF. Cultural, morphological and enzymatic properties of listeria isolated from environmental objects and clinical material. Problems of ecological and medical genetics and clinical immunology. 2001; 4(36): 261-268.
  14. Shapoval VF. Characteristics of factors of pathogenicity of listeria isolated from objects of the external environment and clinical material. Ukrainian Medical Almanakh. 2001; 4(3): 123-129.
  15. Shapoval VF. Epidemiolohichni kharakterystyky infektsiyi listeriozu ta polipshennya metodiv yoho diahnostyky ta profilaktyky [Epidemiological characteristics of listeriosis infection and improvement of methods of its diagnosis and prevention]. Abstr. PhDr. (Med.). K; 2002. 145 p. [Ukrainian]
  16. Shapoval VF. Clinical-epidemiological and immunological features of listeriosis infection in modern conditions. J Clin Exp Med Research. 2001; 32(11): 155-159.