Rapid and sensitive method for the detection of Aeromonas caviae and Aeromonas trota by polymerase chain reaction

Authors


Dr Carl E. Cerniglia Director Division of Microbiology, US Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, U.S.A. (e-mail : CCERNIGLIA@NCTR.FDA.GOV).

Abstract

A 16S rDNA-based polymerase chain reaction (PCR) method was developed for the detection of Aeromonas caviae and Aeromonas trota. These two species were identified from other Aeromonas spp. and closely related species by primers set (AER1 and AER2). The amplified product was 316 bp. The identity of the amplified product was confirmed by DNA–DNA hybridization. Two sets of primers (AER8 and AER9) were used for specific identification of Aer. caviae. Amplifying the 260 bp fragment of 16S rRNA gene region and digesting it with AluI restriction enzyme, yielded 180- and 80-bp fragments. For PCR assay, template DNA was released by mixing equal volumes of homogenized seeded crab meat with Aer. caviae and Chelex 100 (6%) incubated for 10 min at 56°C followed by addition of an equal volume of 0·1% Triton-X-100 and boiled for 10 min. The detection limit was between 50 and 100 cells g−1 of crab meat. This method is very rapid and obviates the need for DNA isolation from complex food matrices and is specific for detecting two Aeromonas species.

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