Bulletin "Veterinary biotechnology"

Veterynarna biotekhnolohiia – Veterinary biotechnology, 2022, 41, 26-34 [in Ukrainian]. https://doi.org/10.31073/vet_biotech41-03

ZHOVNIR A.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MINTCIUK E.P., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TARASOV O.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

 

APPLICATION OF MULTIPLEX PCR FOR THE DETECTION OF TOXIN-PRODUCING CL. PERFRINGENS IN BIOLOGICAL SAMPLES

 Introduction. C. perfringens causes toxico-infectious diseases such as gastroenteritis and acute diarrhea in humans, and their toxins play an important role. This pathogen can produce and release up to 16 various toxins in different combinations. However, four major toxins, namely alpha, beta, epsilon and iota, which are encoded as cpa, cpb, etx and iot genes, respectively, are produced and secreted by C. perfringens isolates, causing intestinal diseases. According to the main toxin profile produced by C. perfringens, this pathogen is classified into five distinct toxinotypes consisting of A, B, C, D and E. Most diseases caused by C. perfringens strains are mediated by the combination of one or more of these toxins

The goal of this study was to determine the molecular toxinotype of C. perfringens isolates, collected in 2019-2021.

Materials and methods. It was used strains and isolates of C. perfringens, stored and maintained at the Institute of Veterinary Medicine. C.perfringens was cultured in Kitta-Tarozzi medium (manufactured at IVM NAAS according to the classical recipe), Wilson-Blair medium (BioRad, India), which was prepared according to the manufacturer’s instructions. DNA was extracted from bacterial cells using a DNA Purification Kit QIAamp® DNA Mini Kit (Cat. No. 51304-Qiagen). Extracted DNA was tested for concentration and purity with a Nanodrop 1000 Spectrophotometer (Thermo Scientific Inc., USA) according to the manufacturer’s instructions. The obtained concentration was 50μg/ml.

Toxin-encoding genes – cpa, cpe, cpb, etx and netB genes were detected and identified using a conventional multiplex PCR assay. Specific primers set, which have previously been described by Chukwu et al. (2016) was used in this study. The PCR products were revealed using electrophoresis in agarose gel at 120 V for 40 min and photographied in transilluminator under UV.

Results of research and discussion. Molecular typing of C. perfringens using multiplex PCR is a fast and effective method of typing toxin-producing isolates of C. perfringens. As a result of the conducted studies, it was established that types A and B were predominant among all isolates (47.8 and 34.7% of all tested isolates, respectively).

All tested isolates of C. perfringens (100%) contained the cpα gene encoding alpha-toxin; 22 isolates of C. perfringens (47.8%) contained cpα and εtx genes, 16 isolates (34.7%) contained cpα and cpe genes. Six isolates (13.04%) were characterized by the presence of cpα, cpe and netB genes. Only three isolates (6.5%) contained genes encoding alpha-, beta- and epsilon toxins. One isolate (2.17%) was characterized by the presence of cpα and cpβ genes.

Conclusions and prospects for further research. Studies have shown that multiplex PCR is rapid and effective approach for C. perfringens typing correlated with traditional methods. Alpha toxin and netB toxin both have critical role in pathogenesis of clostridia-mediated infections The peculiarities of toxine-production genes composition have the potential to be further investigated for the sensitivity and specificity increasing of diagnostic tests.

Kywords: C. perfringens, toxigenic type, PCR, diagnostics, isolate.

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