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

Hwang SH, Kim DE, Sung H, Park BM, Cho MJ, Yoon OJ, Lee DH - PLoS ONE (2015)

Bottom Line: The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR).The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925).The PCR-GO system detected MTB DNA 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%).

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do, 410-769, Republic of Korea; Hematologic Malignancy Branch, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do, 410-769, Republic of Korea.

ABSTRACT
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 the Mycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution from October 2010 to March 2012. To detect MTB in the samples, we performed PCR amplification of the IS6110 DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence was measured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence intensity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTB DNA 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.

No MeSH data available.


Related in: MedlinePlus

Fluorescence signaling after PCR-GO is proportional to the amplicon concentration.Fluorescence in tubes containing 0 (NTC) to 105 copies/reaction of a plasmid encoding the IS6110 sequence is shown. Twenty microliters of a 1 mg/mL stock of GO was added at a final concentration 0.2 mg/mL. The fluorescence images were obtained using the IVIS Lumina Series Ⅲ imaging system. NTC, no-template control.
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pone.0136954.g002: Fluorescence signaling after PCR-GO is proportional to the amplicon concentration.Fluorescence in tubes containing 0 (NTC) to 105 copies/reaction of a plasmid encoding the IS6110 sequence is shown. Twenty microliters of a 1 mg/mL stock of GO was added at a final concentration 0.2 mg/mL. The fluorescence images were obtained using the IVIS Lumina Series Ⅲ imaging system. NTC, no-template control.

Mentions: As shown in Fig 2, the fluorescence intensity displayed gradual increases with increasing concentrations of fluorescent dye-labeled amplicons.


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

Hwang SH, Kim DE, Sung H, Park BM, Cho MJ, Yoon OJ, Lee DH - PLoS ONE (2015)

Fluorescence signaling after PCR-GO is proportional to the amplicon concentration.Fluorescence in tubes containing 0 (NTC) to 105 copies/reaction of a plasmid encoding the IS6110 sequence is shown. Twenty microliters of a 1 mg/mL stock of GO was added at a final concentration 0.2 mg/mL. The fluorescence images were obtained using the IVIS Lumina Series Ⅲ imaging system. NTC, no-template control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556190&req=5

pone.0136954.g002: Fluorescence signaling after PCR-GO is proportional to the amplicon concentration.Fluorescence in tubes containing 0 (NTC) to 105 copies/reaction of a plasmid encoding the IS6110 sequence is shown. Twenty microliters of a 1 mg/mL stock of GO was added at a final concentration 0.2 mg/mL. The fluorescence images were obtained using the IVIS Lumina Series Ⅲ imaging system. NTC, no-template control.
Mentions: As shown in Fig 2, the fluorescence intensity displayed gradual increases with increasing concentrations of fluorescent dye-labeled amplicons.

Bottom Line: The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR).The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925).The PCR-GO system detected MTB DNA 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%).

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do, 410-769, Republic of Korea; Hematologic Malignancy Branch, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do, 410-769, Republic of Korea.

ABSTRACT
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 the Mycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution from October 2010 to March 2012. To detect MTB in the samples, we performed PCR amplification of the IS6110 DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence was measured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence intensity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTB DNA 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.

No MeSH data available.


Related in: MedlinePlus