Secondment to the University of Freiburg

Yu-Ting Kao, NaMeS PhD student, participated in the secondment at the University of Freiburg. The objectives was to start a new project related to droplet microfluidics that can be written in the PhD thesis under the NaMeS project 19 “Droplet Microfluidics to Implement Fast Digital Assays in Centrifugal Microfluidics”. After discussing with supervisors in the University of Freiburg, Yu-Ting Kao decided to develop a novel one-step droplet fluorescence in situ hybridization (FISH) assay integrated on already existed centrifugal microfluidic cartridges in the lab. The goal of this project is for i) universal bacteria detection and enumeration by gene detection method ii) broad spectrum bacterial lysis in droplets, iii) rapid time-to-results, and iv) decreasing manual workload. In this project, Yu-Ting achieved to i) design a universal DNA/LNA hybrid probe to target most of bacteria species, ii) find a universal lysis reagent to lyse both gram positive and gram negative bacteria, iii) integrate the FISH assay to droplets, and iv) detect and enumerate gram negative bacteria in 45 minutes. However, there are still some difficulties that has to be overcome. For example, the time frame of bacterial lysis by the lysis reagents that were used. In this case, it is important to ensure the bacterial lysis does not occur before the bacteria encapsulated into droplets. In addition, the lysis reagent, although can lyse most of bacteria species, suppress the hybridization between targeted rRNA and probes which results in weak fluorescence intensity. Further optimization of the composition of the hybridization buffer and the concentration of the probe needs to be done.

Dates

1 Mar.2019 - 31 Dec. 2020

Location

Freiburg im Breisgau, Germany

Participant

Yu-Ting Kao