• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1887-1894
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• 2007

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.

• Journal of Life Science
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• v.27 no.7
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• pp.746-753
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• 2017

In order to rapidly identify four drums species, Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates, a highly efficient and quick method has been developed using multiplex polymerase chain reaction (PCR) with species-specific primers. Around 1.4 kbp of the mitochondrial COI gene sequences from the four drums species were aligned, and species-specific forward primers were designed, based on the single nucleotide polymorphism (SNP). The optimal conditions for PCR amplification were selected through cross-reactivity, using a gradient PCR method. The PCR results demonstrated species-specific amplification for each species at annealing temperatures between 50 and $62^{\circ}C$. Multiplex species-specific PCR (MSS-PCR) amplification reactions with four pairs of primers were performed for sixteen specimens of each species. MSS-PCR lead to a species-specific amplification of a 1,540 bp fragment in L. polyactis, 1,013 bp in A. nibe, 474 bp in L. crocea, and 182 bp in P. elongates, respectively. The four different sizes of each PCR product can be quickly and easily detected by single gel electrophoresis. The sensitivity of the MSS-PCR of the DNA was up to $0.1ng/{\mu}l$ as a starting concentration for the four different species tested. These results suggest that MSS-PCR, with species-specific primers based on SNP, can be a powerful tool in the rapid identification of the four drums species, L. polyactis, L. crocea, A. nibe, and P. elongates.

• Development and Reproduction
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• v.23 no.4
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• pp.367-375
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• 2019

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

• Korean Journal of Medicinal Crop Science
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• v.13 no.2
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• pp.121-125
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• 2005

An analysis of RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) was performed with three Angelica species (A. gigas Nakai, A. sinensis (Olive.) Diels and A. acutiloba Kitag) in an effort to distinguish between members of these three species. Two arbitrary primers (OPC02, OPD11) out of80 primers tested, produced 17 species-specific fragments among the three species. Eight fragments were specific for A. sinensis, four fragments specific for A. gigas, five specific for A. acutiloba. When primers OPC02 and OPD11 were used in the polymerase chain reaction, RAPD-PCR fragments that were specific for each of the three species were generated simultaneously. Primer OPC02 produced eight species-specific fragments: four were specific for A. sinensis, one for A. gigas, and three for A. acutiloba. Primer OPD11 produced nine speciesspecific fragments: four for A. sinensis, three for A. gigas, and two for A. acutiloba. The RAPD-PCR markers that were generated with these two primers should rapidly identify members of the three Angelica species. The consistency of the identifications made with these species-specific RAPD-PCR markers was demonstrated by the observation that each respective marker was generated from three accessions of each species, all with different origins. We also performed the RAPD-PCR analysis with the dried Angelica root samples that randomly collected from marketed and from the OPC02 primer, obtained a A. gigasspecific band and the band were cloned and sequenced.

• The Korean Journal of Mycology
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• v.44 no.2
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• pp.103-107
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• 2016

To establish a rapid and accurate detection of Phytophthora pinifolia, which is a quarantine pathogenic fungus in Korea, a species-specific primer was developed based on the ras-related protein (Ypt1) gene. Species-specific primer based on the DNA sequences of Ypt1 gene amplified 193 bp polymerase chain reaction (PCR) product for P. pinifolia. The primer pair yielded the predicted PCR product size exactly in testing with target pathogen DNAs, but not from the other 10 species of Phytophthora and 14 species of other phytopathogenic fungi. The primer pair also showed only the species-specific amplification curve on realtime PCR on target pathogen DNA. The detection sensitivity of real time PCR using species-specific primer pair was 10 to 100 times higher than conventional PCR, with 1 to $10pg/{\mu}L$.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.70-76
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• 2011

PCR-based Weissella species-specific detection method was developed to apply for the discrimination of Korean and Chinese kimchi by detecting a Weissella species only found in Korean or Chinese kimchi. PCR primers were designed from the species-specific sequence in the recN gene of each species. The primers allowed the species-specific detection and identification of nine species in the genera Weissella, and were successfully applied to the detection of W. cibaria, W. confusa, W. koreensis, and W. soli in kimchi with 20 ng template DNA. W. cibaria, W. confusa, and W. koreensis were detected from the Korean kimchi samples tested but W. soli was not detected. However, the four species were detected from Chinese kimchi samples. PCR-based W. soli-specific detection could not be perfectly applied as the Chinese kimchi discriminating method but has significance as an approach to evaluate the potential of scientific verification method based on the difference of microbial community.

• Fisheries and Aquatic Sciences
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• v.26 no.11
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• pp.678-688
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• 2023

Flatfish are one of the largest families in the order Pleuronectiformes and are economically important edible marine fish species. However, they have similar morphological characteristics leading to challenges in classifying correctly, which may result in mislabeling and illegal sales, such as fraudulent labeling of processed food. Therefore, accurate identification is important to ensure the quality and safety of domestic markets in Korea. Species-specific primers were prepared from the mainly consumed eleven species of the order Pleuronectiformes. To rapidly identify the 11 flatfish species, a highly efficient, rapid, multiplex polymerase chain reaction (PCR) with species-specific primers was developed. Species-specific primer sets were designed for the mitochondrial DNA cytochrome c oxidase subunit I gene. Species-specific multiplex PCR (MSS-PCR) either specifically amplified a PCR product of a unique size or failed. This MSS-PCR analysis is easy to perform and yields reliable results in less time than the previous Sanger sequencing methods. This technique could be a powerful tool for the identification of the 11 species b the family Pleuronectidae and can contribute to the prevention of falsified labeling and protection of consumer rights.

• International Journal of Oral Biology
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• v.35 no.1
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• pp.21-25
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• 2010

This study was undertaken to develop species-specific forward and universal reverse PCR primers for the detection of Streptococcus sobrinus. These primers target the variable regions of the 16S ribosomal RNA coding gene (rDNA) and their specificity was tested against 10 strains of S. sobrinus strains and 20 different species of oral bacteria using serial dilutions of the purified genomic DNA of S. sobrinus ATCC $33478^T$. Our data show that species-specific amplicons were obtained from all the S. sobrinus strains tested but not from other species. Both direct and nested PCR could detect as little as 400 pg and 4 fg of genomic DNA from S. sobrinus ATCC $33478^T$, respectively. This result suggests that these PCR primers are highly specific and sensitive and applicable to the detection of S. sobrinus.

• Journal of Animal Science and Technology
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• v.50 no.1
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• pp.9-18
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• 2008

Species-specific polymorphisms in chicken, pheasant, turkey, and quail were identified by cloning and sequencing of the immunoglobulin constant domain (IgLC). A set of species-specific primers were then designed on the basis of polymorphisms in the IgLC between species, as well as two additional sets of primers for the cytochrome b and tapasin genes, for the purpose of species identification. Together, the primers successfully distinguished specific species from chicken by species-specific PCR. This simple but unambiguous method may be used to screen avian inter-species germline chimeras, which are valuable models for the conservation of endangered species.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.115-125
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• 1994

In this study, we try to establish the rapid and specific detection system for Salmonella species. The PhoE gene of Salmonella species was amplified with two specific primers, ST5 and ST8c, using PCR. The probe prepared from the amplified PhoE gene was sequenced and applied for Southern blot analysis. After PCR with ST5 and ST8c primers for PhoE gene, DNA bands of expected size(365bp) from 7 different Salmonella species were detected, but not from 12 enterobacteriaceae and 3 gram positive bacteria. PCR was highly sensitive to detect up to 10fg of purified DNA template and to identify Salmonella species with only 320 heat-lysed bacterial cells. The inhibition of PCR amplification from stool specimen was occurred with 50-fold dilution but disappeared over 100 fold dilution of samples. It was confirmed that the PhoE genes were amplified and cloned with over 97% nacleotide sequence homology of PCR products compared with that of S. typhfmurium LT2. The DNA probe derived from S. typhimurium TA 3,000 showed highly specific and sensitive reaction with PCR products of all tested Salmonella species. These results indicate that PCR was rapid and sensitive detection method for Salmonella species and DNA probe prepared from S. typhimurium TA 3,000 was specific to identify PCR products of different Salmonella species.