Development of recombinant positive control for detection of porcine circovirus type 3 by polymerase chain reaction
-
Published:2021-09-28
Issue:3
Volume:7
Page:19-23
-
ISSN:2411-0388
-
Container-title:Journal for Veterinary Medicine, Biotechnology and Biosafety
-
language:en
-
Short-container-title:J. Vet. Med. Biotechnol. Biosafety
Author:
Rudova N. G.1ORCID, Lymanska O. Yu.1ORCID, Bolotin V. I.1ORCID, Stegniy B. T.1ORCID, Solodiankin О. S.1ORCID, Gerilovych А. P.1ORCID
Affiliation:
1. National Scientific Center ‘Institute of Experimental and Clinical Veterinary Medicine’, Kharkiv, Ukraine
Abstract
This work aimed to obtain positive control using recombinant DNA technology for detection by PCR of a new poorly studied pathogen — porcine circovirus type 3. Recombinant positive control was designed using Clone Manager Basic. As a vector in the creation of recombinant control we used plasmid pTZ57R/T, as an insert — a fragment of the gene rep PCV-3 with the length of 418 nucleotide pairs, obtained by classical PCR. Transformation of competent cells of E. coli strain DH5a was carried out by chemical poration, followed by plating on LB-medium with the addition of ampicillin at a final concentration of 100 μg/ml. The selection of E. coli cell colonies was performed by the marker of antibiotic resistance to ampicillin. The presence of a specific insert was checked by PCR with electrophoretic visualization of the results. The developed recombinant positive control can be used for the monitoring of biological samples from pigs for the presence of genetic material PCV-3 using molecular technologies
Publisher
Kharkiv Entomological Society
Subject
Pharmacology (medical),Complementary and alternative medicine,Pharmaceutical Science
Reference31 articles.
1. Bera, B. C., Choudhary, M., Anand, T., Virmani, N., Sundaram, K., Choudhary, B. and Tripathi, B. N. (2020) ‘Detection and genetic characterization of porcine circovirus 3 (PCV3) in pigs in India’, Transboundary and Emerging Diseases, 67(3), pp. 1062–1067. doi: https://doi.org/10.1111/tbed.13463. 2. Caasi, D. R. J., Arif, M., Payton, M., Melcher, U., Winder, L. and Ochoa-Corona, F. M. (2013) ‘A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays’, Journal of Microbiological Methods, 95(2), pp. 229–234. doi: https://doi.org/10.1016/j.mimet.2013.08.017. 3. Camacho, D., Reyes, J., Franco, L., Comach, G. and Ferrer, E. (2016) ‘Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics’ [Clonación de secuencias de alfavirus y flavivirus para uso como controles positivos en el diagnóstico molecular], Revista Peruana de Medicina Experimental y Salud Pública, 33(2), pp. 269–273. doi: https://doi.org/10.17843/rpmesp.2016.332.2101. [in Spanish]. 4. Chan, M., Jiang, B. and Tan, T.-Y. (2016) ‘Using pooled recombinant plasmids as control materials for diagnostic real-time PCR’, Clinical Laboratory, 62(10), p. 1893–1901. doi: https://doi.org/10.7754/Clin.Lab.2016.160114. 5. Chen, J.-M., Guo, L.-X., Sun, C.-Y., Sun, Y.-X., Chen, J.-W., Li, L. and Wang, Z.-L. (2006) ‘A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction’, Biotechnology Letters, 28(22), pp. 1787–1792. doi: https://doi.org/10.1007/s10529-006-9161-0.
|
|