Russian Journal of Parasitology
UDC 619.616.993.192.6
DOI:10.12737/16663
Article history:
Received 16.10.2015
Accepted 24.11.2015
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Dna diagnostics of anaplasmosis in cattle
Samuylenko A. Ya 1 , Gulyukin M. I. 2, Vasilevich F. I.3, Koval'chuk S. N. 4, Glazko T. T. 4, 5, Babiy A. V. 4, Arhipov A. V. 4, Kosovski G. Yu.4
1 All-Russia Research and Technology Institute of Biological Industry, 141142 Moscow region, Shchelkovsky district, village of Biocombinat
2 All-Russian Research Institute of Experimental Veterinary Medicine named after Kovalenko Ya. R., 109428, Moscow, 24-1 Ryasansky Ave.
3 Moscow State Academy of Veterinary Medicine and Biotechnology named after K.I. Skryabin, 109472, Moscow, 23 Academician Skryabin St.
4 FSBI Center of Experimental Embryology and Reproductive Biotechnology, 127422, Moscow, 12 - 4, Kostyakov St., e-mail: info-ceerb@mail.ru
Abstract
Objective of research: The purpose of our research was to develop a DNA diagnostic method for anaplasmosis in cattle.
Materials and methods: Blood samples were obtained from the tail vein with the use of ethylenediaminetetraacetic acid as an anticoagulant. The extraction of DNA was performed with the kit Sorb-M. To analyze the gene msp4 the following sequences belonging to different isolates Anaplasma marginale were used. To select primers the conserved elements of sequences were detected with the server СlustalW2. The specificity of primers was checked by a BLASTN search. The results of polymerase chain reaction (PCR) were estimated using 2% agarose gel electrophoresis. Electrophoresis was performed within 40 minutes at the field intensity 5 V/cm. Fragments of gene msp4 obtained as a results of polymerase chain reaction (PCR) were purified, ligated and cloned in E. coli cells. Transformation was performed using the heat shock method. Search for E. coli colonies containing pGEM-msp4 plasmid was conducted by the PCR method using standard M13 primers with the following analysis of PCR results by electrophoresis.
Target colonies of E. coli were cultured overnight at 37 °С in 2 ml LB medium containing ampicillin in a 100 mcg /ml concentration. Sequencing of received plasmids pGEM-msp4 was carried out by Sanger method and the genetic analyzer Applied Biosystems 3130.
Results and discussion: Development and approbation of primers on the basis of the MSP4 gene of Anaplasma marginale to perform DNA diagnostics of anaplasmosis in cattle by PCR method were described. Due to PCR sensitivity along with the use of primers, it is possible to identify 100 and more gene copies. The conducted experiments confirm the 100% repeatability and reproducibility of the present method.
Keywords: Anaplasma marginale, cattle, diagnostics, PCR.
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© 2015 The Author(s). Published by All-Russian Scientific Research Institute of Fundamental and Applied Parasitology of Animals and Plants named after K.I. Skryabin.
This is an open access article under the Agreement of 02.07.2014 (Russian Science Citation Index (RSCI)) and the Agreemnt of 12/06/2014 (CABI org / Human Sciences section)