Development of a fluorescent 16S rRNA oligonucleotide probe specific to the family Enterobacteriaceae

A fluorescent 16S rRNA oligonucleotide probe was developed specific to the family Enterobacteriaceae. The 16S oligonucleotide target sequence is 5 '-CUCGCGAGAGCAAGCGG-3 ', corresponding to positions 1273 to 1289 on the Escherichia coli 16S rRNA. Specificity of the oligonucleotide probe to the family Enterobacteriaceae was confirmed using representative members of the 20 genera comprising the family and other selected bacteria. Successful application of the probe to an environmental habitat was demonstrated using wastewater samples collected from primary and secondary effluent of a wastewater treatment plant (WWTP). DAPI and EUB338, an oligonucleotide probe specific to the domain Bacteria, were used to enumerate total and prokaryotic cells, respectively, in wastewater samples collected from primary and secondary effluent. The probing efficiency, defined as the number of bacteria enumerated with EUB338 divided by the total number of organisms enumerated with DAPI, in both samples was approximately 94%. The relatively high probing efficiency indicates the rRNA copy number is sufficiently high within bacteria in primary and secondary effluent to ensure a detectable fluorescent image. The origonucleotide probe can potentially be used in the environmental engineering profession as an alternative to the standard coliform test and as a tool to evaluate the effect of upstream processes at a WWTP on disinfection performance.