Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

Diseases of Aquatic Organisms

J. L. Barja¹  A. E. Toranzo¹  C. R. Osorio¹  J. L. Romalde¹ 

关键词: Multiplex-PCR;    Photobacterium damselae;    ureC gene;    16S rRNA gene;    Subspecies discrimination;   

DOI  :  10.3354/dao040177

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspeciespiscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bpand the other of 448 bp, corresponding to internal fragments ofthe 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNAprobe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes thefirst successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operonsequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.

【 授权许可】

Unknown