Real-Time Diagnostics of Anaplasmosis in Cattle by PCR Method

Abstract

The purpose of the research is real-time development of PCR method for diagnostics of anaplasmosis in cattle. Materials and methods. For real time development of primers and fluorescence-labeled probe for PCR msp1α gene sequences 57 isolates Anaplasma marginale available on database Genbank (http://www.ncbi.nlm.nih.gov/genbank/) were used. Conservative areas of msp1α gene were revealed with Сlustal Omega programme (https://www.ebi.ac.uk/Tools/msa/clustalo/). Specificity of primers and probe were checked experimentally in silico using BLASTN programme (http://blast.ncbi.nlm.nih.gov/Blast.cgi) on the animals’ blood samples infected by Anaplasma A. ovis, A. centrale, A. bovis, A. phagocytophilum and A. platys, and sequence analysis of amplicon by Sanger’s method. pGEM-msp1α plasmid designed by us containing msp1α gene fragment with a length of 207 bps was used to assess of sensitivity of the method. Results and discussion. PCR method has been developed in real time mode to detect A. marginale anaplasmosis agent in cattle. Primers and fluorescence-labeled probe have been developed to amplify and detect msp1α gene fragment with a length of 207 bps and PCR conditions have been optimized. Sensitivity of the method allows to detect one copy of msp1а gene copy of А. marginale in analysed DNA sample. Specificity of method allows to differentiate A. marginale from other anaplasma types (A. ovis, A. bovis, A. centrale, A. phagocytophilum, A. platys). The developed method can be used to detect and assess А. Marginale quantitatively in blood samples of infected animals in order to prove the diagnosis as well as to perform epizootological monitoring of anaplasmosis in cattle.