Detection of mutations in Mycobacterium tuberculosis resistant to rifampicin by quantitative PCR: high-resolution melting curve using a single recombinant plasmid as a positive control

• Grant number: 12/17994-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2012
• End date: March 31, 2013
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Mario Hiroyuki Hirata
• Grantee: Camila Rodrigues Gomez
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The RpoB gene, that encodes the ²-subunit of the RNA polymerase, the rifampicin target, is a marker for multidrug resistance. Mutations in this gene, related to rifampicin resistance, are well correlated with resistance to other drugs commonly applied against Mycobacterium tuberculosis (Mtb). Using techniques that facilitates the detection of these mutations reduce the period of time required to cure infected patients, blocks disease transmission, and reduces the treatment cost. Real Time Quantitative PCR followed by High resolution melting (qPCR-HRM) have been used for detecting mutations. This assay consists of DNA amplification in the presence of a fluorescent DNA dye followed by DNA dissociation, which is quantified according to the temperature range. A single base mutation can be identified by comparing the DNA melting curve profile of samples and controls. In a previous study, a segment of 158bp, encompassing the Rifampicin Resistance Determining Region (RRDR) found in the RpoB was amplified, cloned into the pGEM-T and used as control to detect mutations in 54 clinical isolates. The results were highly specific and sensible for detecting mutations when compared with DNA sequencing. The aim of this research is to facilitate the detection of mutation in the RpoB gene by combining plasmid controls and samples in a single tube. The lack of hybridization between non complementary DNA will generate unique melting curve profiles for each mutation studied making the mutation detection easily detected by the machine operator without the need of further analysis. A total of one hundred strains will be evaluated by qPCR-HRM and the results compared to DNA sequencing. Additionally, detection of drug resistance can be coupled with Mtb detection. (AU)