title: Label-free DNA-based detection of Mycobacterium tuberculosis and rifampicin resistance through hydration induced stress in microcantilevers.
abstract: We have developed a label-free assay for the genomic detection of Mycobacterium tuberculosis and rifampicin resistance. The method relies on the quantification of the hydration induced stress on microcantilever biosensors functionalized with oligonucleotide probes, before and after hybridization with specific targets. We have found a limit of detection of 10 fg/mL for PCR amplified products of 122 bp. Furthermore, the technique can successfully target genomic DNA (gDNA) fragments of length >500 bp, and it can successfully discriminate single mismatches. We have used both loci IS6110 and rpoB as targets to detect the mycobacteria and the rifampicin resistance from gDNA directly extracted from bacterial culture and without PCR amplification. We have been able to detect 2 pg/mL target concentration in samples with an excess of interfering DNA and in a total analysis time of 1 h and 30 min. The detection limit found demonstrates the capability to develop direct assays without the need for long culture steps or PCR amplification. The methodology can be easily translated to different microbial targets, and it is suitable for further development of miniaturized devices and multiplexed detection.
authors.affiliation: IMM-Instituto de Microelectrónica de Madrid (CNM-CSIC) , Isaac Newton 8, PTM, E-28760 Tres Cantos, Madrid, Spain.
authors.forename: Carmen M
authors.initials: CM
authors.lastname: Domínguez
authors.forename: Priscila M
authors.initials: PM
authors.lastname: Kosaka
authors.forename: Alma
authors.initials: A
authors.lastname: Sotillo
authors.forename: Jesús
authors.initials: J
authors.lastname: Mingorance
authors.forename: Javier
authors.initials: J
authors.lastname: Tamayo
authors.forename: Montserrat
authors.initials: M
authors.lastname: Calleja
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D015374:Biosensing Techniques / Q000295:instrumentation; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004867:Equipment Design; D006801:Humans; D057230:Limit of Detection; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D012293:Rifampin / Q000494:pharmacology*; D014376:Tuberculosis / Q000175:diagnosis / Q000188:drug therapy* / Q000382:microbiology*
chemical_list: D000904:Antibiotics, Antitubercular; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012293:Rifampin