Rxivist logo

A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

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

Posted 29 Aug 2017
bioRxiv DOI: 10.1101/181974

Here we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high-throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluoresence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co-amplified products with different fluorophores could be simultaneously analyzed to achieve high-throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w/w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.

Download data

  • Downloaded 334 times
  • Download rankings, all-time:
    • Site-wide: 83,683
    • In biochemistry: 2,390
  • Year to date:
    • Site-wide: 132,090
  • Since beginning of last month:
    • Site-wide: 132,887

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide