Development of a rapid identification method for Aeromonas species by multiplex-PCR.
Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.
Sen
Keya
K
Office of Water, Technical Support Center, US Environmental Protection Agency, 26W M.L. King Drive, Cincinnati, OH 43268, USA. sen.keya@epa.gov
D000333:Aeromonas / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D008049:Lipase / Q000235:genetics; D010196:Pancreatic Elastase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D008049:Lipase; D010196:Pancreatic Elastase; D027081:DNA Gyrase