Molecular Characterization of Clinical Isolates to Study rpoB Gene in Mycobacterium tuberculosis Complex
One of the major health problems that can be seen around the world is Anti-microbial resistance and the main cause behind it, is easy availability and higher consumption of medicines. Rifampicin Resistance Tuberculosis (RRTB) occur when rpoB (RNA polymerase) gene in Mycobacterium tuberculosis (MTB) gets mutated and this rpoB gene encodes for beta-subunit of RNA polymerase. Clinical specimens including pulmonary and extra-pulmonary secretion were collected from patients and detected for Tuberculosis (TB) positive cases by targeting mpb64 (Mannose-Binding Protein) gene at 240 base pair. TB positive samples were taken to pre-amplification area for master-mix preparation for Polymerase Chain Reaction (PCR) by targeting specific gene (rpoB) by using forward (5’-CCGCGATCAAGGAGTTCTTC-3’) and reverse primer (3’-ACACGATCTCGTCGCTAACC-5’). Samples were then taken to thermal cycler PCR for amplification of rpoB gene and then amplicons were taken for electrophoresis following the analyzing of amplicons in Electronic UV trans-illuminator system, in which rpoB gene was targeted at 315 base pair to determine if sample is positive for Rifampicin resistance.
The experiment was carried out at Central Molecular Research Laboratory, Department of Biochemistry, Shri Guru Ram Rai Institute of Medical and Health Sciences (SGRRIM&HS), Patel Nagar, Dehradun, Uttarakhand. Patients, who met the criteria of being TB positive cases, were recruited for the drug resistance study. Clinical specimens which includes Pulmonary such as Sputum, Pleural fluid, Bronchiolar alveolar lavage, bronchial secretions and extra pulmonary specimens such as Pus, Urine, Semen, Tissue, Endometrial blood, Cerebrospinal Fluid were considered for the study. Specimens were collected from patients attending Out Patient Department (OPDs) and In Patient Departments (IPDs) of Shri Mahant Indresh Hospital, Dehradun, (Uttarakhand) India. Collected samples were taken for further testing in areas:
Bio-safety cabinet: After collecting the sample, centrifugation process was done for isolating the DNA and the isolation was done by silica column method.
Pre-amplification area: This area was used for master mix preparation for PCR by targeting the specific gene (mpb64 and rpoB at 240 and 315 base pair respectively).
Amplification area: In this area the amplification or multiplication of targeted gene was done by PCR.
Post amplification area: After amplification the product was tested or analysed by using electrophoresis unit.
Resistance towards any anti-tubercular drug, act as an obstacle in the treatment of tuberculosis. The cause for drug resistance of Rifampicin could be patient’s incompliance with their prescription as well as easy availability of drug (Rifampicin) at the drug counter for nose and throat infection. Carelessness in the diagnosis of TB may lead to drug resistance, which can affect the duration of TB treatment. mpb64 gene is not sufficient for the diagnosis of drug resistance in TB, so for this purpose rpoB gene (responsible for drug resistance) is targeted.
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