Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Tuberculosis is still one of the leading human infectious diseases, with 8.7 million new cases reported in 2011 alone. Also, the increasing rate of multidrug-resistant tuberculosis (MDR-TB) and its treatment difficulties pose a serious public health threat especially in developing countries. Resistance to isoniazid and rifampicin, first line antibiotics, is commonly associated with point mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis complex (MTBC). Therefore, the development of a cheap, fast and simple molecular method to assess susceptibility profiles would have a huge impact in the capacity of early diagnosis and treatment of MDR-TB patients. Gold nanoparticles functionalised with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for MTBC and single base mutations associated with antibiotic resistance, namely the characterisation of the three most relevant codons in rpoB gene associated to rifampicin resistance. In this work the Au-nanoprobe approach is extended towards the discrimination of specific mutations within inhA gene which is associated with resistance to isoniazid. Using a multiplex PCR reaction for rpoB and inhA genes, it was possible to assess both loci in parallel, and extend the potential of the Au-nanoprobe method to MDR-TB molecular characterisation with special application in the most frequent genotypes circulating in the Lisbon Health Region.