Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics

Abstract

The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription– polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5′UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5′UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.