Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.
Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.
Schmidt
Marcin T
MT
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland. mschmidt@up.poznan.pl
Olejnik-Schmidt
Agnieszka K
AK
Myszka
Kamila
K
Borkowska
Monika
M
Grajek
Włodzimierz
W
D001422:Bacterial Adhesion; D018938:Caco-2 Cells; D004847:Epithelial Cells / Q000382:microbiology*; D006801:Humans; D052201:Lacticaseibacillus rhamnosus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*