Development of the test kit for the differential diagnostics of african and classical swine fever by RT-PCR in real time
Journal Title: Бюлетень "Ветеринарна біотехнологія" - Year 2017, Vol 31, Issue
Abstract
Introduction. African swine fever (ASF) and Classical swine fever (CSF) are two transboundary contagious viral swine diseases with high mortality rates and huge economic impacts. ASF cannot be differentiated from CSF by either clinical presentation or post mortem examination, therefore in the event of a suspected outbreak, differential diagnosis of the two diseases is essential.The goal of the work is to develop specific primers to the target DNA and RNA fragments of ASFV and CSFV respectively and to determine the optimal conditions of the amplification.Materials and methods. Pathological and biological materials with ASFV and CSFV, cell cultures containing closely related viruses were used in the study. «RIBO-Sorb» (AmpliSens) was used for DNA extraction. Amplification was performed using Rotor-Gene 6000 («QIAGEN Hilden»). The nucleotide sequence of primers were designed through Primer Express software (Applied Biosystems).Results of research and discussion. The OIE primers for real-time PCR were used for detection of ASF DNA. The primers to the target 72 bp fragment of CSFV 5’ UTR region for detection of CSF RNA were selected using Primer Express. With the purpose to control DNA/RNA extraction and exclude the presence of PCR inhibitors, an internal control (IC) was constructed and specific primers with fluorogenic probe were also designed.Primer annealing temperatures and optimal protocol of PCR were optimized.The specificities of the primer pairs were determined with pathogens of PCV2, PRRS and Aujeszky's disease. The cross-reactions with closely related DNA and RNA viruses were not observed.Conclusions and prospects for further research. Specific primer pairs and TaqMan probes for the conserved regions of ASFV DNA, CSFV RNA and IC were selected. The conditions of amplification were determined for the duplex real-time RT-PCR for the differential diagnosis of ASF and CSF. In further investigations the validation of the developed test kit will be carried out.
Authors and Affiliations
Svetlana Mandygra, Larysa Muzykina, L. Ishchenko, Anna Kovalenko, Ihor Halka, Nikolai Sytjuk, Serghiy Nychyk, V. Spyrydonov
Keywords
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- EP ID EP266488
- DOI 10.31073/vet_biotech31-12
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