Bioinformatics Techniques for Developing Molecular Detection Methods for the HIV-1 Gag Gene

Abstract

The HIV-1 Gag gene, which plays an essential role in HIV replication, can be detected accurately using qRT-PCR. The quality of qRTPCR analysis is determined by the primers and probes used for DNA amplification. This research aims to use bioinformatics techniques to design primer pair sequences and qRT-PCR probes for HIV detection using the HIV-1 Gag gene. HIV-1 Gag gene sequences were obtained from HIV-1 isolates and serotypes, downloaded from the National Center for Biotechnology Information (NCBI) GenPeptd nucleotide database. Sequences were then examined using the ClustalW algorithm of the Bioedit sequence alignment editor version 7.2.5.0. through gene alignment using multiple sequence alignment (MSA) with conserved regions. The primer pair sequences of the Gag-HIV 1 gene were obtained, namely, forward 5'-CAGTACAATGTGCTTCCACAGGG-3 and reverse 3'-CGGGATAGAGATTCAGTCTAGG-5' with the probe sequence 5'-GGATCACCAGCAATATTTCAGGGAACG-3'. The primer sequence has a length of 23 bases (forward), 22 bases (reverse), GC content of 52% (reverse), 50% (forward), and the same forward and reverse melting temperature (Tm) of 66°C. The probe sequence is 27 bases long, with a GC content of 48% and a Tm of 67.3°C. No hairpin loops and dimers were formed in the primer pair or probe, and the gag gene had 100% homology with HIV-1. It was concluded that the primer and probe pair sequences met the requirements and could be used to amplify the HIV-1 Gag gene using qRT-PCR.