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

Yanan Du,Bin Shi,Wei Shi,Wenjing Wu,Binan Zhao,Ruili Hu,Fangfang Ren,Wanjing Zhang,Xiao Zhao,Xiaoxia Zhang,Xiaorui Fan,Yan Xu,Kai Zhao,Yangyang Wu,Huanzhen Zhao,Qi Zhang

doi:10.1038/s41598-018-36981-1

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.

Highlights

The increases in the number of biomolecular samples that need to be analyzed generate a great demand for a high-throughput detection method
Each pair of primers specific to a target sequence was labeled with a specific fluorophore that did not interfere with other fluorophores in the same reaction set (Fig. 1)
The results suggested that four genetically modified (GM) maize could be detected simultaneously starting from 0.5% level, which was lower than levels prescribed by EU regulation and was consistent with singleplex analysis results

Summary

Introduction

The increases in the number of biomolecular samples that need to be analyzed generate a great demand for a high-throughput detection method. DNA microarray is a high-throughput approach used for analyzing complex nucleic samples that have limited feasible availability due to the complicated procedure and expensive consumption Poor linearity is another weakness of DNA microarray that limits its use in quantitative analysis[17]. Developing a novel qualitative, quantitative and high-throughput method for detecting multiple biological genes is of great importance. We constructed and validated a novel high-throughput detection method based on combining emulsion PCR and fluorescence spectrophotometry (EPFS) for qualitative, quantitative and high-throughput detection of multiple biological genes. The new method uses the primer pairs labeled with different fluorophores, so that the multitarget DNAs in one mixed sample could be amplified in a single reaction set; and qualitatively and quantitatively analyzed on Infinite M1000 PRO. The EPFS method we developed provides a new approach for qualitative, quantitative and high-throughput analysis of multitarget DNAs for a broad range of biological samples

Methods

Results

Conclusion