ABS352FD01209

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation, storage, and DNA extraction, techniques that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is a rapid, isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried, reaction-ready enzyme pellets that can be used to analyze crude specimens at the point of need. This project aimed to develop a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces. An anonymized human fecal sample from a deceased Caucasian female with a medical history of hypoglycemia and hypothyroidism was obtained from Analytical Biological Services Inc. (donor ID# ABS352FD01209). The composition of the fecal microbiome was independently characterized using the gold standard method, 16S rRNA sequencing. A QIAamp DNA Stool mini kit was used to isolate DNA from the fecal sample. SeqWright Genomic Services (Houston, Tx) generated and validated a library of 300-bp amplicons (MiSeq Reagent kit v3, MS-102-3001) using the extracted DNA and a universal primer pair spanning the 16S rRNA variable 4 region(Caporaso et al., 2011). The relative abundance of A. muciniphila was then calculated by comparing the number of reads classified as A. muciniphila to the total number of bacterial reads. Sequence-based and RPA-based estimates of A. muciniphila were then compared to asses RPA's accuracy in quantifying the relative abundance of A. muciniphila.