Primer design and optimization for the amplification of RV1337 genes among Mycobacterium tuberculosis isolates obtained from the Mycobacteriology Laboratory at MakBRC

Abstract

Worldwide, Mycobacterium tuberculosis causes more deaths than any other single microbial agent with approximately one-third of the world’s population being infected with this organism. It is estimated that nearly 2 billion people about one fourth of the world’s population are infected with Mycobacterium tuberculosis. Every year, about 10 million people develop TB disease and 1.6 million people die of it (CDC, 2019). The RV1337 genes are genes that encode a rhomboid protease 2 protein in mycobacteria, such as Mycobacterium tuberculosis (Kateete et al., 2010). The RV1337 genes are also involved in biofilm formation and resistance to some antibiotics that target DNA gyrase (Kateete et al., 2012). However, RV1337 gene regulation, substrate specificity, and contribution to drug resistance and pathogenesis are poorly understood implying the need of such a research study. To design and optimize specific primers for the amplification of the RV1337 genes among Mycobacterium tuberculosis isolates obtained from the Mycobacteriology Laboratory at MAKBRC. In silico primer design was heuristically executed by means of a free trial version of a commercial software program called SnapGene version 7.2.0.0(GSL Biotech LLC Co.) using the FASTA format of the RV1337 gene sequence copied from GenomeNet online database KEGG T0015:RV1337.The designed primer pair was optimized by a series of gradient PCR techniques used to determine the optimal annealing temperature of 68ºC. The modified CTAB method was used for DNA extraction whose yield was quantified by Nanodrop spectrophotometry. Conventional PCR was used to amplify the target gene from the extracted DNA using the designed primer pair at 68ºC. The amplicons from the different PCRs were all quality controlled by agarose gel electrophoresis and then UV visualization to confirm the required amplifications. A band amplification of 720bps which correlates with the size of the RV1337 gene was obtained upon PCR amplification using the designed primer pair.