SYBR green real time‐polymerase chain reaction as a rapid and alternative assay for the efficient identification of all existing Escherichia coli biotypes approved directly in wastewater samples

Abstract

Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm‐blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses SYBR green real time‐polymerase chain reaction (RT‐PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT‐PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT‐PCR analysis can encourage the testing of a higher number of samples. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1106–1113, 2012

Detection and Occurrence of Indicator Organisms and Pathogens

“…The species T4 (associated with pathogenic strains) accounted for 66.7% of positive samples in the Puzih River. Investigators also determined that physical characteristics (like temperature) and microbiological characteristics (like total coliforms) were associated with the occurrence of Acanthamoeba.Another study showed that real time-PCR (RT-PCR) is a more effective and practical approach than the conventional methods, which cannot identify E. coli serotype O157:H7(Chetta et al, 2012). 54 wastewater samples were analyzed using developed primers, and thus detection, of three E. coli-specific genes -HNS (DNA-binding transcriptional dual regulator), Allan (allantoin permease), and CadC (DNA-binding transcriptional activator) -that were highly conserved between 33serotypes.…”

Reclaimed water use pilot scale practices in the Catalonian region. Viability quantitative PCR for microbial water quality monitoring

“…Melting temperature is defined as the temperature at which 50% of the oligonucleotide-target duplexes remain hybridized (Mackay et al, 2007). SYBR Green has been successfully used in real time PCR to detect microorganisms in wastewater and reclaimed water (Chetta et al, 2012;Dong et al, 2010;Ferrer et al, 2012;González et al, 2010;Nayak and Rose, 2007).…”