Use of fluorescence quantitative polymerase chain reaction (PCR) for the detection of Escherichia coli adhesion to pig intestinal epithelial cells

• Autorzy: Dai C.H. , Gan L.N. , Qin W.U. , Zi C. , Zhu G.Q. , Wu S.L. , Bao W.B.
• Języki publikacji: EN

Abstrakty

EN

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.

• Wydawca: -
• Czasopismo: Polish Journal of Veterinary Sciences
• Rocznik: 2016
• Tom: 19
• Numer: 3
• Opis fizyczny: p.619-625,fig.,ref.

Twórcy

autor

Dai C.H.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

autor

Gan L.N.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

autor

Qin W.U.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

autor

Zi C.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

autor

Zhu G.Q.

• College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China

autor

Wu S.L.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

autor

Bao W.B.

• College of Animal Science and Technology, Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China

Bibliografia

• Atshan SS, Nor Shamsudin M, Sekawi Z, Lung LT, Hamat RA, Karunanidhi A, Mateg Ali A, Ghaznavi-Rad E, Ghasemzadeh-Moghaddam H, Chong Seng JS (2012) Prevalence of adhesion and regulation of biofilm-related genes in different clones of staphylococcus aureus. J Biomed Biotechnol 2012: 976972.
• Bertschinger HU, Moon HW, Whipp SC (1972) Association of Escherichia coli with the small intestinal epithelium. I. Comparison of enteropathogenic and nonenteropatho-genic porcine strains in pigs. Infect Immun 5: 595-605.
• Candela M, Perna F, Carnevali P, Vitali B, Ciati R, Gionchetti P, Rizzello F, Campieri M, Brigidi P (2008) Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production. Int J Food Microbiol 125: 286-292.
• Hohmann A, Wilson MR (1975) Adherence of enteropathogenic Escherichia coli to intestinal epithelium in vivo. Infect Immun 12: 866-880.
• Imberechts H, Bertschinger HU, Stamm M, Sydler T, Pohl P, De Greve H, Hernalsteens JP, Van Montgu M, Lintermans P (1994) Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or post-weaning diarrhoea. Vet Microbiol 40: 219-230.
• Li X, Deng LX, Wu GX, Cao Y, Fan B, Zhao J, Han Q, Chen SZ, Gao X, Huang N (2010) Effects of HMGN2 on bacterial adhesion to Hela Cells. Medical Journal of West China 22: 788-792.
• Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods 25: 402-408.
• Melkebeek V, Rasschaert K, Bellot P, Tilleman K, Favoreel H, Deforce D, De Geest BG, Goddeeris BM, Cox E (2012) Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response. Mucosal Immunol 5: 635-645.
• Nataro JP, Kaper JB (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11: 142-201.
• Schmittgen TD (2001) Real-time quantitative PCR. Methods 25: 383-385.
• Shi XH, YM, Zhou X, Chen X, Fan DL (2015) Relationship of surface properties of silicone rubber with bacteria adhesion. J Third Mil Med Univ 37: 1921-1925.
• Tawakoli PN, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C (2013) Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clin Oral Investig 17: 841-850.
• Turner SM, Scott-Tucker A, Cooper LM, Henderson IR (2006) Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiol Lett 263: 10-20.
• Wall SJ, Edward DR (2002) Quantitative reverse transcription-polymerase chain reaction (RT-PCR): A comparison of primer-dropping, competitive, and real-time RT-PCRs. Anal Biochem 300: 269-273.
• Wu SL, Yuan ZW, Ju HP, Huang XG, Hua JD, Zhou GY, Wang JY, Xie KZ, Chen GH (2007) Polymorphisms of the FUT1 gene M307 locus in post-weaning Sutai breed piglet and resistance to F18 fimbrial Escherichia coli in vitro. Chinese Journal of Preventive Veterinary Medicine 29: 783-787.
• Wu ZC, Dong WH, Liu Y, Yang JS, Zhu GQ, Wu SL, Bao WB (2014) Attak Experiment and Phenotype Analysis of Meishan Piglets by E. coli F18 Strain. Acta Veterinaria Et Zootechnica Sinica 45: 1608-1615.
• Yu L, Wu J, Zhu GQ (2012) Heat-labile enterotoxin enhances etec adherence to porcine small intestine epithelial cell line IPEC-J2. Chinese Journal of Animal Infectious Diseases 20: 29-35.
• Zhang HY, Wang XB, Zhao XM, Wang YB, Chen LY, Xia PA, Cui BA (2010) Radix Isatis Polysaccharides to Inhibit Adhesion of Pathogenic E. coli to Cell. J Microbiol 30: 61-63.
• Zhang W, Zhao M, Ruesch L, Omot A, Francis D (2007) Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US. Vet Microbiol 123: 145-152.

Identyfikatory

DOI

10.1515/pjvs-2016-0077