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A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets.

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.

SUBMITTER: Du Y

PROVIDER: S-EPMC6336815 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets.

Du Yanan Y Zhao Xiao X Zhao Binan B Xu Yan Y Shi Wei W Ren Fangfang F Wu Yangyang Y Hu Ruili R Fan Xiaorui X Zhang Qi Q Zhang Xiaoxia X Zhang Wanjing W Wu Wenjing W Shi Bin B Zhao Huanzhen H Zhao Kai K

Scientific reports 20190117 1

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 flu ...[more]

PMID: 30655629

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Project description:Illicit drugs are a global health problem, since both their acute and chronic consumption have negative impacts on the drug user's health. Drug checking facilities are receiving growing interest as they allow drug users to chemically analyze their product prior to consumption to assess the presence of adulterants or other non-expected substances. Such harm reduction programs allow the reduction of the risks associated with drug consumption without encouraging it. In particular, the emergence of new psychoactive substances (NPS) emphasizes the risk for the population increasing the diversity and the lability of illicit drugs on the market. Analytical developments are required to catch up with this rapid evolution and reduce the potential harm caused by such consumption. In this study, we developed a matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) strategy for the high-throughput qualitative and quantitative analysis of drug checking samples. The use of online-based m/z cloud library for untargeted compound search improved the ability to identify unknown compounds. Sixty-seven drug checking samples were analyzed using this analytical strategy, allowing the detection of 10 designer drugs and several classical drugs of abuse (mainly cocaine and MDMA) as well as adulterants and contaminants. The results were then compared with routine analyses of the same samples using conventional approaches showing similar performance while removing the use of chromatographic separation thus resulting in a significant reduction of the time required for sample preparation and analysis. This study enlightens the potential of MALDI-HRMS as a high-throughput approach allowing to speed-up up to six times the identification and quantification of substances enabling to catch the fast changes on the drug of abuse market. This strategy could be an interesting alternative analytical approach, allowing better prevention and harm reduction for drug users.

Dataset Information

A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets.

Publications

A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets.

Similar Datasets

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