Australian researchers are fine-tuning a new, more efficient genotyping platform for pathogen screening, leveraging microsphere bead technology to carry out multiplexed PCR assays to simultaneously test multiple gene variants without the need for unique internal probes customized for each target.
Featured on the cover of the January 1 edition of Analytical Biochemistry, the method outpaces a standard TaqMan real-time PCR system, which has a maximum of five signal channels, by a factor of 20, supporting up to 100 detection channels at a time (based on the current library of color-coded microspheres).
“With optimization of the quantity and quality of these microspheres along with the density of oligonucleotides on them, high-level multiplexing is a realistic goal,” wrote the authors, led by Ross Barnard, PhD, biotechnology program director and affiliate professor in the Queensland Alliance for Agriculture and Food Innovation at the University of Queensland’s School of Chemistry and Molecular Biosciences.
After DNA is extracted from samples, it’s mixed with the color-coded Luminex microspheres. “Each color corresponds one-to-one with a particular target pathogen,” explained Dr. Barnard. A PCR reaction is then carried out in a tube containing the color-coded beads. “A fluorescent signal develops on those beads on which a positive reaction takes place. After the reaction, the microspheres are separated in a capillary sorting machine.”
Both the color code identifying the pathogen and the green signal on each microsphere can be measured at the same time; the stronger the green signal, the more of the corresponding pathogen is present in the sample. “The time required for one run is approximately the same as existing techniques, but the ability to do many more reactions at once means that many potential pathogens could be tested for at once, in a single run,” observed Dr. Barnard, who holds patents on the technology.
Proof of principle for the technology has been established with three targets—influenza A and two genes in Neisseria meningitidis (meningococcus). Next, Dr. Barnard and his colleagues plan to test multiple pathogens, including Campylobacter coli and Campylobacter jejuni genotypes (of which there are many) and other disease targets.
The ongoing development of the technology will be supported by Australia’s Poultry Cooperative Research Centre and the University of Queensland.