September 1, 2016.
Probiotics are live microorganisms, like bacteria and yeast, which are thought to provide health benefits when ingested. Large manufacturers produce blends of probiotics for supplementing the diets of agriculturally important animals. Commercial probiotic products are labeled with the number of cells present at the time of manufacture. Our assignment was to develop an assay which can be used to quantify the number of specific lactic acid bacteria present in certain probiotic products, thus verifying the number reported on the product labels. This quantification process involves isolating genomic DNA (gDNA) from the given samples and then running the DNA through a quantitative polymerase chain reaction (qPCR). We optimized gDNA isolation by 1) treating these gram-positive bacteria with lysozyme and proteinase K, and 2) increasing the length of cell disruption by bead beating (FastPrep). Results showed that gDNA yields were improved by longer FastPrep treatments, but not by enzyme treatments. Quantitative polymerase chain reactions (qPCR) on isolated gDNA allow researchers to make copies of a short target DNA sequence and monitor its amplification in real time through the use of a fluorescent probe. Using this method, we created standard curves for four species of bacteria which allowed us to correlate cell quantity to threshold cycle. These standard curves allow us to quantify the number of each bacteria present in probiotic mixes of unknown composition.
Bioinformatics | Biotechnology
California Polytechnic State University (Cal Poly SLO)
This material is based upon work supported by the National Science Foundation through the Robert Noyce Teacher Scholarship Program under grant #1340110. Any opinions, finding, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The research was made possible by the California State University STEM Teacher Researcher Program in partnership with California Polytechnic State University San Luis Obispo.