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.

Direct Detection of OXA-48 Carbapenemase Gene in Lysate Samples through Changes in Mechanical Properties of DNA Monolayers upon Hybridization

“…27 Given the good results achieved working with the static mode, as a first approach we have attempted to detect the plasmid measuring the difference in the surface stress generated with hydration after hybridization. For details, see references 27,33 . Figure 2 shows the representative surface stress curves obtained with a relative humidity cycle for the microcantilevers sensitized with the mixture of ssDNA probes (black line in a) and b)) and after incubation with purified DNA from OXA-48 + KP isolates (green line in a)), which contain the plasmid harboring the blaOXA48 gene; and OXA-48 -KP isolates (red line in b)).…”

“…26 However, they have not been established yet as alternative techniques to the most used methods. 13 Microcantilevers have proven to be highly sensitive label-free biosensors for microbial DNA detection [27][28][29] and have the potential for developing instruments suitable for both laboratory and point-of-care testing. Here we report the labelfree and PCR-free detection of blaOXA48 gene using total extracted DNA from carbapenem resistant Klebsiella pneumoniae (KP) cultures.…”

Advances in microfluidics in combating infectious diseases

“…(Fang et al, 2012). Recently, a label-free assay for detection of DNA of M. tuberculosis and related drug resistance has been designed (Domínguez et al, 2015). This platform makes use of the quantification of hydration induced stress on micro-cantilever functionalized oligonucleotide probes before and after hybridization with specific nucleic acid targets.…”

Detection methods for centrifugal microfluidic platforms

“…For example, if an added molecular layer contracts compared to the underlying material, the cantilever will bend towards the molecular layer. This measuring principle has been used for example; to detect prostate-specific antigen (Wu et al, 2001), to study antibiotics binding to drug resistant "super bugs" (Ndieyira et al, 2008), and to detect Mycobacterium tuberculosis and rifampicin resistance (Domínguez et al, 2015). Also, bacteria responses to antibiotics have been studied by recording fluctuations in deflection of cantilevers coated with bacteria (Longo et al, 2013).…”