Rapid Identification of Mutations in the rpoB of Mycobacterium tuberculosis using Helicase Dependent Amplification (HDA) and Amplified Ligated Loop (ALL) DNA Assay

Abstract

The immediate diagnosis of multi-drug resistant tuberculosis (MDRTB) in the Philippines is limited by the use of culture-based detection methods that usually take 4-8 weeks. Amplified ligated loop (ALL) assay, a DNA-based assay able to detect alterations in the genome, coupled with thermophilic helicase dependent amplification (tHDA) was used to distinguish mutations in codon 526 of the rifampicin resistance determining region (RRDR) of M. tuberculosis. The study aims to develop a simple and fast technique that would be able to detect rifampicin resistance of M. tuberculosis in ten (10) isolates. The DNA of ten confirmed M. tuberculosis isolates were amplified using tHDA and were subjected to ALL DNA assay. A positive result was marked by the fluorescence of the nitrocellulose paper under UV light. Nine out of ten LCP samples did not have mutations at the 526 codon while LCP 89 had a point mutation of C->T as was detected by the ALL assay. The assay results matched with the DNA sequences of the isolates.