Access Type

Open Access Thesis

Date of Award

January 2015

Degree Type

Thesis

Degree Name

M.S.

Department

Nutrition and Food Science

First Advisor

Diane Cabelof

Abstract

Lactic acid bacteria (LAB) and Bifidobacterium are the most common types of microbes used as probiotics. They are present in the human gastrointestinal tract and have a significant influence on our health and well-being. Microbiota plays an important role in host metabolism and provides a natural defense mechanism against invading pathogens. This experiment was focusing on establish a method to detect the gastrointestinal tract microbiota, either by fecal or colonic tissue DNA extraction.

The experiment comparing 2 types of DNA extraction; ZR Fecal kit and DNAzol direct. DNAzol direct was easy to use but was not suitable for long term DNA storage, hence the sample need to be extract fresh when needed. The extracted DNA, when amplified with G.Lacto genus specific primer show 99% match with sequence of Lactobacillus reuteri in database. For primer sensitivity test, G.Bifid1 genus specific primer could detect DNA up to a dilution of 10-10, or approximately 10 ag of DNA. For species specific primers, BIA could detect DNA up to a dilution of 10-8, or 1 fg of DNA. Both BiLon and BiBre primer could detect DNA up to a dilution of 10-7, or 10 fg of DNA. The PCR product of G.Bifid1 primer amplified against ATCC bacteria DNA:-resulted in 99% match with all 3 bacteria sequences (B. breve, B. Adolescentis, and B. Longum). When using BiBre and BIA primers amplified against B. breve and B. infantis ATCC DNA, the sequences were 100% similar to each species in bacteria database. For BiLon primer, the results yield 96% match.

The difference in age of the mice could play an important role in the presence of bacteria detected in fecal samples, due to unequally delayed development of the young mice's intestinal ecosystems. The presence of bacteria in feces might not represent the type or quantity of bacteria that inhabitant the colon. One challenge that needs to be overcome in recovery of DNA is finding the lysis method that could penetrate peptidoglycan, the heavily cross-linked structure in gram-positive bacteria cell wall. Also find the method to verify the development of the pathophysiological changes, that are claimed to be responsible for the variation in bacterial levels in the GI tracts of the mice, would make the results more accurate. Further large-scale research in animals and humans, especially randomized controlled trials, is warranted.

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