RESEARCH ARTICLE Simple Detection of the IS6110 Sequence of Mycobacterium tuberculosis Complex in Sputum, Based on PCR with Graphene Oxide

Graphene oxide (GO) has proven to be a satisfactory DNA-sensor platform for applications in enzyme-free signal amplification, fluorescence-based amplification, and nanoparticle-based platforms because of its excellent electrical, thermal, and optical properties. In this study, we designed a novel platform for the fluorescence detection of biomolecules, using a fluorescent dye-labeled primer and GO.We applied this system for the detection of the IS6110 insertion sequence of theMycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution fromOctober 2010 to March 2012. To detect MTB in the samples, we performed PCR amplifi-cation of the IS6110DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence wasmeasured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence inten-sity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110DNAwere in good agree-ment with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTBDNA in 23 of 25 RQ-PCR-positive sputum samples (92.0%; 95%CI, 75.0–98.0%), but not in 29 of 29 RQ-PCR-negative sputum samples (100%; 95%CI, 88.1– 100.0%). These results indicate the utility of the PCR-GO system in molecular diagnostics