Development of random amplified polymorphic DNA-based specific polymerase chain reaction primers for the detection of the histamine-producing bacterium Photobacterium damselae subsp. damselae

Abstract

Photobacterium damselae subsp. damselae is a marine bacterium that has the ability to produce histamine. In this study, 26 P. damselae subsp. damselae strains were isolated from Scomber australasicus (mackerel). These strains were assayed for histamine production using a histamine immunoassay test kit. The results demonstrated that all strains were histamine-producing bacteria, and most of them were strong histamine producers, yielding more than ≥1000 ppm histamine. To develop a polymerase chain reaction (PCR)-based method to detect P. damselae subsp. damselae, the random amplified polymorphic DNA (RAPD) method was used to design the specific primers. The 330-bp RAPD fragment present in all the isolated P. damselae subsp. damselae strains was cloned and sequenced. Specific primer sets were designed from this RAPD fragment. These primers were highly specific to P. damselae subsp. damselae and could distinguish it from P. damselae subsp. piscicida. The sensitivity of these primers specific for P. damselae subsp. damselae was approximately 1 fg. For the fish sample system, the specific primers enabled the detection of n × 100 colony forming units P. damselae subsp. damselae cells per gram of fish sample if a 16-h enrichment step was performed prior to PCR. This PCR-based assay of the RAPD fragment could provide a sensitive and specific method for detecting P. damselae subsp. damselae.