Genomic fingerprinting of Escherichia coli strains using repetitive sequence based polymerase chain reaction
Nikam, Amar (2004) Genomic fingerprinting of Escherichia coli strains using repetitive sequence based polymerase chain reaction. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Repetitive element PCR (rep-PCR) uses outward facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphism of rep-PCR amplification products can serve as strain specific molecular fingerprints. In this study, capability of repetitive BOX elements, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) was tested to detect genetic diversity among Escherichia coli strains recovered from autopsied poultry birds showing different pathological conditions. The DNAs from 52 avian E.coli isolates were extracted and used to amplify BOX, ERIC and REP sequences. DNA from avian strains produced 1-11 bands by BOX-PCR, 2-13 bands by ERIC-PCR, where as 1-13 bands by REP-PCR. All these primers showed good discriminating power. BOX-PCR produced 23 different products where as ERIC-PCR and REP-PCR produced 26 and 25 products, respectively. All bandswere showing 100 per cent polymorphism. Dendrograms based on different patterns revealed extensive genetic diversity among avian strains. Three major clusters were formed by BOX-PCR and ERIC-PCR showing less than 25 per cent similarity where as two major clusters were formed by REP-PCR showing less than 25 per cent similarity. Network analysis of these patterns showed highly complex network suggesting intense heterogeneity among E.coli strains. Network obtained by BOX primer was most complex where as network obtained by REP-PCR was least complex among these three primers. The results obtained were compared with Serotypes, Biotypes and SSCP analysis. No correlation could be established but those isolates that could not be analyzed or discriminated by above typing techniques were easily analyzed and discriminated by rep-PCR analysis. These results suggest intense heterogeneity or genetic diversity among E.coli strains studied. Also, rep-PCR has discriminating capacity that could improve the studies needed to understand genetic makeup of E.coli strains.
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