Development of Rapid and High-Throughput Genetic Fingerprinting Assay for the Identification of Specific Intestinal Microbes

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There is an increasing awareness that our microbiota plays an important role in a variety of pathologicaldisorders including obesity, diabetes, Parkinson's disease, multiple sclerosis, and colorectal cancer.Personalized medicine will evolve to include assays for the presence or absence of particular microbes incomplex samples (e.g. stool). Most microbes are uncultivable and current genomic sequencing techniques arehigh resolution but slow and expensive. There will be an increasing need for targeted, inexpensive, and rapidculture-free methods to identify particular microbes from a variety of patient and environmental samples.MassArray technology is used to identify genetic polymorphisms for genetic testing. MassArray involvesthe selective PCR amplification of as many as 40 different loci (multiplex PCR), where specific variants(mutations) are subsequently identified by mass spectrometry (MALDI-TOF). Arctic Diagnostics Inc.(ArcticDx, Toronto) has extensive experience in the use of MassArray, performing ~100,000 genetic tests onpatients over the past 5 years to identify risk alleles for macular degeneration, cystic fibrosis, and gestationaldiabetes. Given recent evidence that microbiota play a role in each of these diseases, ArcticDx is motivated toexpand the application of their MassArray to identify specific microbial "fingerprints" in patient samples.Toward this end Arctic wishes to establish a collaboration with Dr. William Navarre (University ofToronto), a microbiologist who has spent the past three years developing tools to culture and analyzemicrobiota samples from both humans and mice. We have developed a clear and technically feasible plan todetermine if MassArray technology can be employed to simultaneously identify the presence or absence of upto 20 specific microbes from complex (mouse stool) samples. This will involve the development of multiplexprimer sets specific to individual microbial species and troubleshooting the PCR, MALDI-TOF, andcomputational parameters necessary to achieve acceptable levels of specificity and selectivity.