HIGH THROUGHPUT REAL-TIME PCR FOR DETECTION OF MICROBIAL COMMUNITIES WITHIN THE DISTRIBUTION NETWORK

Abstract

Ensuring the accessibility to safe drinking water is a major challenge being faced by Pakistan, where water-borne ailments are on a continuous rise, owing to inadequate sanitation and poor water management. Conventional techniques for water quality monitoring have low detection efficiency and only detect the microbial concentrations of 102-103 CFU/ml within a water sample. Escherichia coli and Salmonella typhimurium are the enteric pathogens often associated with outbreaks of water-borne illnesses in humans. However, owing to low sensitivity and methodological limitations of traditional treatment methods, these may go undetected or poorly quantified. The aim of present study was to determine the concentrations of Escherichia coli and Salmonella typhimurium in a lab-scale distribution network, using the Quantitative Polymerase Chain Reaction (qPCR), involving SYBR Green chemistry. Isolation of gene sequences for, SLT-I (Shiga like toxin I) and invA gene was achieved using the explicitly designed primer pairs' specific to Escherichia coli and Salmonella typhimurium, respectively. The qPCR analysis lead to enumeration of target bacteria from as little as 3.5 PI and 1.0 PI of template DNA acquired from drinking water spiked with Escherichia coli and Salmonella typhimurium. It was observed that optimized chlorine disinfection dosage of 1.0 mg/L of chlorine, worked more effectively in case of Escherichia coli as compared to Salmonella typhimurium. The amplification efficiency for the two target genes was determined using absolute and relative methods of quantification. It was observed that the adapted assay for the two species worked with different efficiencies. Moreover, optimizing reaction conditions specific to target species, in terms of DNA quality and concentration, yielded greater amplification efficiency. It was concluded that if adapted for water