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, 9 (1), 184

A Novel Emulsion PCR Method Coupled With Fluorescence Spectrophotometry for Simultaneous Qualitative, Quantitative and High-Throughput Detection of Multiple DNA Targets

Affiliations

A Novel Emulsion PCR Method Coupled With Fluorescence Spectrophotometry for Simultaneous Qualitative, Quantitative and High-Throughput Detection of Multiple DNA Targets

Yanan Du et al. Sci Rep.

Abstract

We constructed and validated a novel emulsion PCR method combined with fluorescence spectrophotometry (EPFS) for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets. In a single reaction set, each pair of primers was labeled with a specific fluorophore. Through emulsion PCR, a target DNA was amplified in droplets that functioned as micro-reactors. After product purification, different fluorescent-labeled DNA products were qualitatively analyzed by the fluorescent intensity determination. The sensitivity and specificity of the system was examined using four kinds of genetically modified (GM) maize. The qualitative results revealed high specificity and sensitivity of 0.5% (w/w). In addition, the quantitative results revealed that the absolute limit of detection was 103 copies, showing good repeatability. Moreover, the reproducibility assays were further performed using four foodborne pathogenic bacteria to further evaluate the applicability of the system. Consequently, the same qualitative, quantitative and high-throughput results were confirmed with the four GM maize. To sum up, the new EPFS system is the first analytical technology of this kind that enables simultaneous qualitative, quantitative and high-throughput analysis of multiple genes.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic of EPFS method. Primer pairs are labeled with specific fluorophores in the same reaction set, and template fragments are amplified and labeled with the same fluorophore in the minute aqueous droplets through the emulsion PCR. The purified amplicons tagged with different fluorophores was detected for qualitative, quantitative and high-throughput analysis.
Figure 2
Figure 2
Specificity of the singleplex EPFS assay. (a) The specificity assay for detection of BT176 GM maize. The forward primer of BT176 were labeled with FAM to amplify different DNA samples, and FAM-labeled amplicons were analyzed. (b) The specificity assay for detection of GA21 GM maize. The forward primer of GA21 were labeled with HEX to amplify different DNA samples and HEX-labeled amplicons were analyzed. (c) The specificity assay for detection of NK603 GM maize. The forward primer of NK603 were labeled with ROX to amplify different DNA samples and ROX-labeled amplicons were analyzed. (d) The specificity assay for detection of TC1507 GM maize. The forward primer of TC1507 were labeled with NED to amplify different DNA samples and NED-labeled amplicons were analyzed. (e) The specificity assay for detection of IVR gene of maize. The forward primer of IVR gene were labeled with CY5 to amplify different DNA samples and CY5-labeled amplicons were analyzed. RFU: relative fluorescence units; PC: positive control; NTC: no template control.
Figure 3
Figure 3
Multiplex specificity assay for detection of four GM maize simultaneously using EPFS method in a single reaction. Four pairs of fluorescent-labeled primers were used to amplify four event-specific genes of four GM maize. Four fluorescent-labeled amplicons were analyzed to evaluate the multiplex specificity of the EPFS method. FAM: labeled BT176 amplicons; HEX: labeled GA21 amplicons; ROX: labeled NK603 amplicons; NED: labeled TC1507 amplicons; RFU: relative fluorescence units.
Figure 4
Figure 4
Sensitivity of the multiplex EPFS assay for GM maize detection. Multiplex EPFS assay were performed on a series of DNA solution with a final relative content of 10%, 5%, 1%, 0.5% and 0.1% (w/w). The column with slash means negative, which was lower than the threshold. FAM: labeled BT176 amplicons; HEX: labeled GA21 amplicons; ROX: labeled NK603 amplicons; NED: labeled TC1507 amplicons; RFU: relative fluorescence units.
Figure 5
Figure 5
The EPFS assay was used to quantitatively analyze the GM maize. (a) Four sets of labeled primers targeted amplicons at event-specific gene of four different GM maize. (b) When run in a multiplex reaction, it is obvious that the amplification efficiencies of the four reactions are approximately the same, with similar x-intercepts indicating roughly similar sensitivity. RFU: Relative fluorescence units.

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