Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2018, 32, 565-576 [in Ukrainian]. https://doi.org/10.31073/vet_biotech32(2)-69

UKHOVSKYI V.V.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MUZYKINA L.M.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., GALKA I.V.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SPYRYDONOV V.G.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TSARENKO T.M.2, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., ANTONIK I.I.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., PYSKUN O.O.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., PYSKUN A.V.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SHEVCHENKO T.V. 3, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

1 Institute of Veterinary Medicine of NAAS

2 Bila Tserkva National Agrarian University

3 National Academy of Agrarian Sciences of Ukraine

DEVELOPMENT AND VALIDATION OF REAL TIME POLYMERASE CHAIN REACTION FOR DETECTION DNA OF PATHOGENIC LEPTOSPIRA

Introduction. Currently the polymerase chain reaction (PCR) development and use are conducted for the detection of the genetical and pathological characteristics of Leptospira and leptospirosis diagnostics. Due to this method, it is possible to detect small amounts of Leptospira’s DNA in clinical and environment samples, and for identification of Leptospira cultures as well.

The goal of the work was to develop and validate the DNA protocol for detection of pathogenic Leptospira by the PCR in real time and to optimize the conditions for the amplification reaction.

Materials and methods. To analyze the nucleotide sequences, which encodes the outer membrane lipoprotein LipL 32 synthesis, the sites of pathogenic Leptospira genome were collected from the GenBank on-line database. The composition of the reaction mixture and the temperature parameters of amplification were determined by using DNA from 7 reference strains of pathogenic Leptospira: 493 Poland, Kabura, Perepelicyni, Pomona, Moskva V, Hond Utrecht IV, M 20. The sensitivity of this protocol was studied using DNA-extract from reference pathogenic strain of Leptospira M 20 (serogroup Icterohaemorrhagiae). The specificity of this method was tested on the strains of microorganisms as follow: saprophytic species Leptospira biflexa, strain Patoc 1; Chlamydophila psittaci; Mycoplasma hyopneumoniae; Brucella abortus; Actinobacillus lignieresii; Salmonella cholerae suis; Salmonella dublin; Escherichia coli; Staphylococcus aureus; Streptococcus zooepidemicus; Pasteurella multocida; Fusobacterium necrophorum; Clostridium perfringens; Clostridium septicum.

Results of research and discussion. The results of the development and validation of the method of PCR real-time for the detection of pathogenic Leptospira DNA by international guidelines were revealed. It was established that the developed primer hybridization system showed pronounced activity against DNA template at 45ºС and the concentration of magnesium ions in the reaction mixture of 1,5 mM/microliter. Presented parameters confirmed the sensitivity, specificity and limits of detection techniques. Conclusion and prospects further research. Specific primers LipL 32 F/R were designed on the base of conservative sites analysis of the outer membrane lipoprotein gene Lip L 32 nucleotide sequences of the pathogenic Leptospira. The developed protocol for the detection of pathogenic Leptospira DNA is specific, accurate and provides detection of pathogenic Leptospira DNA in samples with 4000 cells/ml. This protocol will propose for livestock farms to monitoring and control of the spread of leptospirosis.

Keywords: Leptospira, DNA, PCR real-time, amplification protocol, optimization.

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