A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets.
Sci Rep
; 9(1): 184, 2019 01 17.
Article
de En
| MEDLINE
| ID: mdl-30655629
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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Spectrométrie de fluorescence
/
ADN
/
Réaction de polymérisation en chaîne
/
Tests de criblage à haut débit
Type d'étude:
Diagnostic_studies
/
Qualitative_research
Langue:
En
Journal:
Sci Rep
Année:
2019
Type de document:
Article
Pays d'affiliation:
Chine