PCR amplification of species specific sequences of 16S rDNA and 16S–23S rDNA intergenic spacer region for identification of Streptococcus phocae

Institut für Tierärztliche Nahrungsmittelkunde, Professur für Milchwissenschaften, Justus-Liebig-Universität Giessen, Ludwigstr. 21, 35390 Giessen, Germany.
Microbiological Research (Impact Factor: 2.56). 02/2008; 163(2):132-5. DOI: 10.1016/j.micres.2006.01.017
Source: PubMed


Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.

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Available from: José Francisco Fernández-Garayzábal, Dec 15, 2014
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    • "However, it has been widely shown that the internal spacer region (ISR) between the 16S and 23S rRNA genes is more variable between bacterial species than ribosomal genes themselves in both sequence and length (Barry et al., 1991; Hassan et al., 2003; Osorio et al., 2005). Species-specific primers derived from these sequences have also been reported (Kong et al., 1999; Lee et al., 2002; Hassan et al., 2008). In this study, we sequenced the ISR from T. soleae and designed speciesspecific primers, targeting both the 16S rRNA gene and ISR region, for its identification and detection by PCR. "
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    ABSTRACT: The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.
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    ABSTRACT: In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.
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    ABSTRACT: The sensitivity of 4 published primer pairs for the detection of Streptococcus phocae strains was evaluated. Primer sets cpn60-F and -R and sodA-F and -R correctly identified S. phocae. Correct identification was also achieved with the primer pairs cael-cae2 and PX1-PX2, but using the reverse complementary version of both reverse primers (caeVQ2 and PXVQ2, respectively). Among the 4 PCR protocols with pure and mixed cultures, the primer pair PX1-PXVQ2 provided the highest level of sensitivity for S. phocae (10(2) and 10(4) cells per PCR tube), and detection was 10- to 100-fold higher than the other 3 primer pairs. When the cael-caeVQ2 and PX1-PXVQ2 PCR protocols were applied to different seeded Atlantic salmon tissues (spleen, kidney and liver), the detection limit achieved was 5.1 x 10(5) to 6.4 x 10(7) CFU g(-1), and the lowest sensitivity detected was 1.18 x 10(6) S. phocae per tube (which corresponds to 6.4 x 10(7) CFU g(-1)) in spleen samples using PX1-PXVQ2. In the kidney samples seeded with S. phocae strains, regardless of the primer set used, the PCR sensitivity was the same (7.31 +/- 1.5 x 10(6) CFU g(-1)). In addition, the nested PCR assay using the primer pair PX1-PXVQ2 improved the sensitivity of detection of S. phocae by at least 100 times compared to the first round PCR, not only in mixed and pure suspensions, but also in experimentally seeded fish tissues. The picked tissues that allowed the easiest detection of S. phocae were the liver, kidney and spleen, respectively. Thus, the nested PCR approach is an important tool for the rapid and reliable diagnosis of streptococcosis due to S. phocae.
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