Identification of oligonucleotide primers targeted at the 16S-23S rDNA intergenic spacers for genus- and species-specific detection of aeromonads

Abstract

Aeromonads occur widely in coastal waters and wastewater; and have been associated with a wide variety of gastrointestinal infections in humans. The prevalence of Aeromonas spp. in the aquatic environment has been recognized as a potential health risk, and some countries have adopted aeromonad counts as an additional indicator of water quality. In view of the above, we have attempted to develop some species-specific primers for the rapid detection of these organisms by PCR. The 16S-23S rRNA intergenic spacer regions (ISR) of seven Aeromonas species - Aeromonas hydrophila, A. caviae, A. enteropelogenes, A. trota, A. jandaei, A. veronii and A. schubertii were amplified by the Polymerase chain reaction using primers complementary to conserved regions of the 16S rRNA and 23S rRNA genes. The ISR amplimers were cloned into plasmid vectors and sequenced. Analysis of the ISR sequences showed that aeromonads contain two types of polymorphic 16S-23S spacers - ISR(Glu) and ISR(IA). The ISR(Glu) contains the tRNA gene for glutamate while ISR(IA) contains the tRNA genes for isoleucine and alanine. Multiple alignment of representative ISR(Glu) sequences from the seven species revealed several major regions of variability. These regions were used to design Aeromonas genus-specific and species-specific primers for PCR. The specificity of the PCR primers were validated using total DNA prepared from various Aeromonas spp. and bacterial species of other genera. Results also showed that the method can be applied efficiently for routine monitoring of aeromonads in seawater Copyright (C) 1999 Elsevier Science Ltd.