RESUMEN
More and more Single Nucleotide Polymosrphisms of interest among pathogenic organisms are described with the advent of Whole Genome Sequencing but WGS approach is still too expensive, time consuming, and relying on bioinformatical means that are not available in many developing countries. This study presents a low-cost reverse hybridization line probe technique for detecting SNPs in Mycobacterium tuberculosis. The proposed test is able to detect mutations in the RRDR of rpoB gene in M. tuberculosis with specificity and sensitivity of 98% and 100%, respectively and for an average cost of less than 3 per sample. The technique proved efficient not only on pure DNA samples extracted from culture isolates but also on crude extracts from clinical samples. The flexibility of the platform allows to get it transformed to any kind of test detection, hence, building a bridge between rich countries performing SNP discovery and countries with high burden that can target these SNPs on the collected samples.
Asunto(s)
Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Hibridación de Ácido Nucleico/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
While minisatellites are usually typed using capillary sequencers or qiaplex systems in developed countries, many low-resource regions cannot afford it. We propose an optimized agarose gel electrophoresis method to genotype Mycobacterium tuberculosis species complex minisatellites in their standardized format (24 MIRU-VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon sizes whatever the repetition number of each locus. This method performs well both on DNA extracts of good quality and on thermolysates while reducing workload and reagents costs.