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Research Journal of Microbiology
Year: 2007  |  Volume: 2  |  Issue: 2  |  Page No.: 163 - 169

Evaluation of Polymerase Chain Reaction for Direct Detection of Escherichia coli Strains in Environmental Samples

Safa A. Sherfi, Hamid A. Dirar, Badr E. Hago, Mohamed E. Ahmed, Hassan A. Musa, Hassan Abu Aisha and Imadeldin E. Aradaib    

Abstract: The potential of the Polymerase Chain Reaction (PCR), as a means of detecting Escherichia coli (E. coli) DNA in suspected environmental samples, was studied. Using a pair of outer primers P1 and P2, selected from uidA gene, which encodes E. coli glucuronidase, the PCR-based assay resulted in amplification of a 486 base pair (bp) PCR product. E. coli strains from different environmental sources including recycled and drinking water as well as stagnant water were detected by this nested PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gel. The sensitivity of the PCR assay was 100 fg of bacterial DNA with ethidium bromide-stained agarose gels. Using a pair of internal (nested) primers P3 and P4, the nested PCR produced a 186 bp PCR product. The nested PCR increased the sensitivity of the PCR assay by 1,000 times and specific PCR products were detected from 0.1 fg of bacterial DNA. Amplification product was not detected when the nested PCR-based assay was applied to DNA from other related bacteria including, Salmonella, Pseudomonas and Proteus or nucleic acid-free water. Application of this nested PCR-based assay to environmental samples resulted in direct detection of E. coli DNA from sewage water, tap water, drinking water at Shambat Campus, University of Khartoum, Sudan. This nested PCR-based assay should provide a rapid, sensitive and specific assay for direct detection and quantification of E. coli in environmental samples suspected to contain the organism.

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