title: Use of Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Escherichia coli Adhesion to Pig Intestinal Epithelial Cells.
abstract: An efficient and accurate method to test Escherichia coli (E. coli) adhesion to intestinal epithelial cells will contribute to the study of bacterial pathogenesis and the function of genes that encode receptors related to adhesion. This study used the quantitative real-time polymerase chain reaction (qPCR) method. qPCR primers were designed from the PILIN gene of E. coli F18ab, F18ac, and K88ac, and the pig β-ACTIN gene. Total deoxyribonucleic acid (DNA) from E. coli and intestinal epithelial cells (IPEC-J2 cells) were used as templates for qPCR. The 2-ΔΔCt formula was used to calculate the relative number of bacteria in cultures of different areas. We found that the relative numbers of F18ab, F18ac, and K88ac that adhered to IPEC-J2 cells did not differ significantly in 6-, 12-, and 24-well culture plates. This finding indicated that there was no relationship between the relative adhesion number of E. coli and the area of cells, so the method of qPCR could accurately test the relative number of E. coli. This study provided a convenient and reliable testing method for experiments involving E. coli adhesion, and also provided innovative ideas for similar detection methods.
authors.forename: C H
authors.initials: CH
authors.lastname: Dai
authors.forename: L N
authors.initials: LN
authors.lastname: Gan
authors.forename: W U
authors.initials: WU
authors.lastname: Qin
authors.forename: C
authors.initials: C
authors.lastname: Zi
authors.forename: G Q
authors.initials: GQ
authors.lastname: Zhu
authors.forename: S L
authors.initials: SL
authors.lastname: Wu
authors.forename: W B
authors.initials: WB
authors.lastname: Bao
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D007413:Intestinal Mucosa / Q000166:cytology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013552:Swine