Bulletin "Veterinary biotechnology"

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Veterynarna biotekhnolohiia– Veterinary biotechnology, 2023, 43, 35-43 [in Ukrainian]. https://doi.org/10.31073/vet_biotech43-04

ZHOVNIR O.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MINTSIUK E.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SAVCHENIUK M.2, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TARASOV O.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

 1Institute of Veterinary Medicine of the NAAS
2Bila Tserkva national agrarian university

 

DEVELOPMENT OF A TEST KIT FOR THE DETECTION OF C. SEPTICUM BY REAL-TIME POLYMERASE CHAIN REACTION

Introduction. According to published data, the disease caused by C. septicum can occur both alone and in association with other pathogens of the Clostridium group (Clostridium chauvoei, Clostridium sordellii, Clostridium novyi type A), the coccus group and P. aeruginosa. The alpha-toxin gene is highly conserved and is the main target gene for species identification and simultaneous determination of toxin production. The use of molecular genetic methods, such as polymerase chain reaction, allows conducting rapid detection of the pathogen in any sample with high specificity.

The goal of the work. To study the sensitivity and specificity of a set of primers for the detection of C. septicum by real-time polymerase chain reaction.

Materials and methods. As reference samples, the strain and pathogenic field isolates of C. septicum and C. perfringens, C. chauvoei, C. sordellii, C. novyi microorganisms stored in the museum of the Institute of Veterinary Medicine of the National Academy of Sciences of Ukraine were used.

For the study, we used a set of primers for PCR-rt, which consisted of primers and a FAM probe, the design of which was developed by the authors of the article based on the sequence of the csa gene of the reference strain of C. septicum NCTC547 (GenBank D17668).

PCR was performed in a thermocycler (Bio-Rad, S-1000, USA) in a total reaction volume of 25 μL. Reaction volume of 25 μL, containing 17 μL of PCR master mix (Taq 2X Master Mix, NEB, USA), 5 μL of target DNA, 3 μL of primer mixture (containing 20 pmol/mL of primers and 10 pmol/mL of probe).

To detect amplification products, the threshold method was used – the value of the threshold cycle of the reaction Ct (Threshold cycle) was determined. The results of the analysis were considered reliable if the Ct value of the FAM channel was less than or equal to 35 (Ct≤35), the Ct value of the negative control channel was absent, the Ct value of the JOE channel of all tested samples (amplification of target gene fragment) was less than or equal to 35 (Ct≤35).

The results were processed by descriptive statistics using the R package (www.rproject.org)

Results of research and discussion. The limit of detection of the validated method was found to be about 100 genome copies/pathogen DNA reaction.

According to the results shown, the calculated validated standard deviation (SD) for five amplification replicates of each of the six plasmid dilutions was lower than the maximum acceptable level of acceptable SD for the method (SDv≤0.5). The values of the coefficients of variation (% CV) during the experiment were also lower than the acceptable value of the coefficient of variation for the method (CVv). The data obtained indicate a high convergence of the results of pathogen DNA detection.

The detection thresholdΔOD >+0.3 obtained as a result of the study is sufficient for reliable detection of even small amounts of the pathogen.

The analytical specificity was defined as the ability to distinguish the target DNA site from the nucleic acids of other pathogenic Clostridia. Pathological material from cattle containing C. septicum (positive biological material) and bacterial culture suspensions were used to determine the specificity.

Thus, the overall specificity of the PCR-RT test for the target gene of C. septicum was 100% (95% Wilson’s confidence interval: 96.8 to 99.9%), and the sensitivity for samples with 100 genome copies or more was 100% (95% Wilson’s confidence interval: 99.3 to 99.9%).

Conclusions and prospects for further research. 1. The primer set used for PCR-rt, consisting of primers and a FAM probe, the design of which was developed by the authors, based on the sequence of the csa gene of the reference strain of C. septicum NCTC547 (GenBank D17668), has high sensitivity, 100% specificity and allows detection of target DNA in various types of biological material.

2. The overall specificity of the PCR-RT test for the target gene of C. septicum was 100% (95% Wilson’s confidence interval: 96.8 to 99.9%), and the sensitivity for samples with 100 copies of the genome or more was 100% (95% Wilson’s confidence interval: 99.3 to 99.9%). The detection threshold ΔOD >+0.3 obtained in the study is sufficient for reliable detection of pathogen amounts of 100 genome copies and above in the reaction.

The research results will be used to create a diagnostic test system for the detection of clostridia in pathological material.

Keywords: qPCR, diagnostics, primers, DNA, C. septicum.

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