Full Length Research Paper

Development of a competitive PCR assay for the quantification of total Escherichia coli DNA in water

Omar Kousar Banu, Barnard Tobias George* and Jagals Paul

Water and Health Research Unit, University of Johannesburg, Republic of South Africa.

*Corresponding author. E-mail: tgbarnard@uj.ac.za. Tel: +2711 5596324. Fax: +2711 5596329.

Accepted 23 November, 2009

   Abstract

Standard health-related microbial water testing relies on the culturability of Escherichia coli (E. coli) to estimate their numbers. Competitive PCR (c-PCR) offers the potential to estimate the E. coli level of a water source without culturing. The aim was to investigate the use of c-PCR reaction to detect and quantify, without prior enrichment, Escherichia coli in water samples. The E. coli malate dehydrogenase Mdh house-keeping gene was modified and used as an internal control and competitor DNA for the c-PCR. E. coli cell concentration equivalents ranging from 20 to 2 x 10⁴ cells ml^-1 could be quantified with the c-PCR. Fifty-three water samples from various sources were tested with the DNA extraction and c-PCR protocol. Due to PCR inhibition E. coli Mdh gene copies could only be determined for 20 of the 53 samples (38%). Of the 20 samples tested 15% gave comparable results for competitive PCR and culturable E. coli numbers; 55% obtained higher values with competitive PCR and 30% obtained higher values with the culture based experiments. The c-PCR successfully estimated E. coli numbers that gave comparable results with the culture based microbiological data obtained.

Key words: Competitive polymerase chain reaction (c-PCR), waters, Escherichia coli, internal standard.