Design and optimizing of primers used to detect Rv2094c gene in mycobacterium tuberculosis species H3RV following molecular analysis of PCR at the molecular laboratory of MakCHS
Abstract
Mycobacterium tuberculosis is one of the well-known respiratory pathogens for human tuberculosis which persists as a persistent public health threat and statistical models have it documented that on a global scale, Tuberculosis is the second leading infectious disease among the human population after covid-19 above HIV/AIDS and about 1.3 million people have died from TB sine 2022 including those with HIV/AIDS estimated to about 167000 people. According to the recent records, it is estimated that about 10.6 million people suffered from tuberculosis by 2021 which demonstrated an increase of 4.5% as per 2020 and 1.6 million people died due to this same disease including about 187000 HIV positive people according to reports the World Health Organization and in East Africa, with over 23% of new cases and 31% of TB related deaths which is utmost contributed by the heavy burden of HIV/AIDS prevalence which is reflected in the 20% of the new TB cases that are among people living with HIV/AIDS in the region. Drug resistant TB inclusive of both Rifampicin resistant and multi drug resistance is on a steady growth pace affecting over 450,000 and 77000 people in the region respectively. Objective: This study is aimed at designing and optimizing primers for the Rv2()94c gene detection in mycobacterium tuberculosis species H37RV strain during molecular analysis of PCR at the Genomics. Molecular Biology and Immunology laboratory at College of Health Sciences in Makerere University. Methods: It is an experimental qualitative study that employs SnapGene software to design primers for the Rv2094c gene and the designed primers will be ordered and purchased from Macrogen, Europe. Then optimized using Gradient PCR, visualized under Agarose Gel electrophoresis obtain the right annealing temperature basing on the strongest band. The MTB DNA will be extracted using Modified CTAB and quantified using NanoDrop spectrophotometer 2000. Then a conventional PCR is run and then visualization the PCR amplicons using Agarose Gel electrophoresis in order to obtain a band amplification of about 272basepairs.