Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

Bal Hari Poudel,Hemanta Kumari Chaudhary,Mitesh Shrestha,Prakash Chaudhary

doi:10.3126/ijasbt.v5i1.17020

Abstract

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

Highlights

Tuberculosis (TB) is regarded as a historical disease, which has a long and continuing record of causing worldwide morbidity and mortality especially in developing countries, including Nepal (Chadha, 2009)
For detection of mutation in rpoB gene, the amplification-refractory mutation system (ARMS) primers employed was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3′-OH terminus of the primer
Control forward primer and common reverse primer were used in each of the samples, along with codon specific primer, for amplification of 537 bp region of rpoB gene serving as an internal control to avoid false negative results

Summary

Introduction

Tuberculosis (TB) is regarded as a historical disease, which has a long and continuing record of causing worldwide morbidity and mortality especially in developing countries, including Nepal (Chadha, 2009). Infection by the bacillus Mycobacterium tuberculosis leads to the chronic disease with aerial mode of transfer between people (Zaman, 2010). The nature of symptoms depends upon the site where the bacterium inhabits. In the cases of pulmonary TB, it may cause symptoms, such as chronic cough, pain in the chest, haemoptysis, weakness or fatigue, weight loss, fever, and night-sweats (Zaman, 2010). When the M. tuberculosis organism develops resistance to at least Isoniazid and Rifampin, considered as the most effective medication against tuberculosis, they are referred to as Multidrug-resistant tuberculosis. The emergence of primary drug resistance in a patient who has never before been treated for tuberculosis would be due to infection by drug-resistant bacilli. Secondary (acquired) drug resistance arises in a patient, who initially had drugsusceptible M. tuberculosis, through inappropriate or inadequate treatment, noncompliance to a prescribed regimen or poor absorbance of prescribed medication (Gangadharam, 1993)

Methods

Results

Conclusion