Development of quantitative PCR method for Escherichia coli detection in human stool samples

Abstract

The gut microbiome is a complex community of microorganisms that reside in the gastrointestinal tract of humans and animals. This diverse ecosystem includes Escherichia coli (E. coli), which are not just present, but are essential in maintaining health by aiding digestion, synthesising vitamins such as Vitamin B12 and K, and protecting against pathogens. E. coli is a versatile bacterium with various strains, some of which are harmless commensals. E. coli contributes significantly to the microbiome's balance, a normal gut inhabitant. However, pathogenic strains can cause severe foodborne illnesses, and is linked to other chronic conditions. Researchers who study the gut microbiome often start by extracting DNA from faecal samples. Ensuring the purity and integrity of the DNA is not just important, but crucial for downstream applications, such as absolute quantification. One such technique used in the lab is quantitative PCR (qPCR), which allows for precisely quantifying specific DNA sequences. This method is particularly useful in quantifying the absolute abundance of bacteria in faecal samples, such as E. coli, providing invaluable insights into the gut microbiome. In this thesis I established a protocol for DNA extraction from stool samples and attempts to quantify E. coli