B.L. Bueno, F.G. Oliveira, G.K. Lima, A.A. Fonseca Júnior, T.C. Kassar, R.J.F. Câmara, R.C. Leite, J.K.P. Reis
Published: August 07, 2018
Genet. Mol. Res. 17(3): GMR18027
DOI: https://doi.org/10.4238/gmr18027
Cite this Article:
B.L. Bueno, F.G. Oliveira, G.K. Lima, A.A.Fonseca Júnior, T.C. Kassar, R.J.F. Câmara, R.C. Leite, J.K.P. Reis (2018). One-Step RT-qPCR assay for detection and quantification of equine infectious anemia virus in-vitro. Genet. Mol. Res. 17(3): GMR18027. https://doi.org/10.4238/gmr18027
About the Authors
B.L. Bueno, F.G. Oliveira, G.K. Lima, A.A. Fonseca Júnior, T.C. Kassar, R.J.F. Câmara, R.C. Leite, J.K.P. Reis
Corresponding Author
J.K.P Reis
Email: jenner@ufmg.br
ABSTRACT
Equine infectious anemia virus (EIAV) infection often results in an initial febrile response, followed by recurrent cycles of the disease and finally, a prolonged asymptomatic period. These variations in clinical signs are due to a number of factors, including virus strain, equid species and differences in susceptibility among animals. As a consequence of the close relation between viral strain and disease, studies about in-vitro replication and fitness of EIAV in macrophages, which are the target cells of the virus, depend on accurate measurement of viral load throughout the infection period. We developed a method to quantify EIAV in-vitro using a one-step RT-qPCR system from a control RNA synthesized for this purpose. Designed primers amplified a 520 base pair fragment from the gag gene region that was inserted into a pGEM-T Easy Vector plasmid and propagated in Escherichia coli DH5-α. The bacteria with the construct were propagated and sufficient quantities of the template DNA were produced. The RNA was synthesized in-vitro from the plasmid linearization product and was used for standardization of a one-step RT-qPCR system with a minimum detection limit of 10 to 15 molecules. The efficiency of the reaction was 101%, with r2 equal to 0.997. This new method can be used for the determination of virus titer in EIAV replication studies in-vitro.
Key words: EIAV, RNA, synthesis, viral load.