[Journal of Microbiological Methods] Multiple pathogen biomarker detection using an encoded bead arr
We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.
Prem Kumar Periyannan Rajeswaria, Lovisa M. Soderberga, Alia Yacoubb, Mikael Leijonb, Helene Andersson Svahna, Haakan N. Joenssona, , a Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, Tomtebodavägen 23A, 171 65 Solna, Sweden b Department of Microbiology, National Veterinary Institute (SVA), Ulls väg 2B, SE 751 89 Uppsala, Sweden Received 8 March 2017, Revised 19 April 2017, Accepted 19 April 2017, Available online 20 April 2017 Show less http://doi.org/10.1016/j.mimet.2017.04.007