• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.280-289
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• 2021

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

• Animal Systematics, Evolution and Diversity
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• v.26 no.1
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• pp.15-19
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• 2010

The Russian ratsnake, Elaphe schrenckii, is found in Russia, China, and Korea, and is considered to be an endangered species by the Ministry of Environment in South Korea. Due to habitat loss and use in oriental medicine, their population has been severely decimated. In South Korea, two subspecies of E. schrenckii has been defined according to body color: E. s. schrenckii (blackish) and E. s. anomala (yellow-brownish). Molecular genetic studies on Elaphe schrenckii are very scarce and the taxonomy of Elaphe schrenckii subspecies is uncertain. From the present study, we attempted to identify the genetic differences of these two subspecies using species-specific microsatellites developed from the genomic library of E. schrenckii. Nine polymorphic loci were tested on 19 individuals from E. s. schrenckii (n=10) and E. s. anomala (n=9) in South Korea. The mean number of alleles was 3.78 in E. s. schrenckii and 4.11 in E. s. anomala. The average expected heterozygosity was 0.542 and 0.511 in E. s. schrenckii and E. s. anomala, respectively. We found a lack of genetic structure between two subspecies ($F_{ST}=0.016$) and no genetic discrimination between two subspecies was found. Based on the present findings by microsatellites, two subspecies can be considered as one species, E. schrenckii. However, further investigations on taxonomical status using mitochondrial and nuclear DNA sequences need to be performed and morphological & ecological data should be revised. The genetic markers should benefit future studies of the endangered species of other Elaphe species for the study of genetic diversity and potential conservation management.

• Mass Spectrometry Letters
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• v.5 no.4
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• pp.110-114
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• 2014

The method of direct mass spectrometry profiling is reliable and reproducible for the rapid identification of clinical isolates of bacteria and fungi. This is the first study evaluating the approach of MALDI-TOF mass spectrometry profiling for rapid identification of carbapenemase-resistant enterobacteriaceae (CRE). Proof of concept was achieved by the discrimination of CRE using MALDI Biotyper MS based on the protein. This profiling appears promising by the visual observation of consistent unique peaks, albeit low intensity, that could be picked up from the mean spectra (MSP) method. The Biotyper MSP creation and identification methods needed to be optimized to provide significantly improved differences in scores to allow for subspecies identification with and without carbapenemases. These spectra were subjected to visual peak picking and in all cases; there were pertinent differences in the presence or absence of potential biomarker peaks to differentiate isolates. We also evaluated this method for potential discrimination between different carbapenemases bacteria, utilizing the same strategy. Based on our data and pending further investigation in other CREs, MALDI-TOF MS has potential as a diagnostic tool for the rapid identification of even closely related carbapenemases but would require a paradigm shift in which Biotyper suppliers enable more flexible software control of mass spectral profiling methods.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.788-797
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• 2001

The usefulness of three PCR methods were evaluated for the epidemiological typing of Staphylococcus aureus: an enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic element PCR (REP-PCR), and 16S-23S intergenic spacer PCR (ITS-PCR). The analysis was performed using a collection of S. aureus strains comprised of 6 reference and 79 isolates from patients with various diseases. Among the 85 S. aureus strains tested, 6 references and 6 isolates were found to be susceptible to methicillin, whereas the remaining 73 isolates were resistant to it. PCR methods are of special concern, as conventional phenotypic methods are unable to clearly distinguish among methicillin-resistant S. aureus (MRSA) strains. The ability of the techniques to detect different unrelated types was found to be as follows: ERIC-PCR, 19 types; REP-PCR, 36 types; and ITS-PCR, 14 types. On the basis of combining the ERIC, REP, and ITS fingerprints, the 85 S. aureus strains were grouped into 56 genetic types (designated G1 to G56). The diversities for the 85 S. aureus strains, calculated according to Simpson\`s index, were 0.88 for an ERIC-PCR, 0.93 for a REP-PCR, and 0.48 for an ITS-PCR, and the diversity increased up to 0.97 when an ERIC-PCR and REP-PCR were combined. The above discrimination indices imply that the genetic heterogeneity of S. aureus strains is high. Accordingly, this study demonstrates that DNA sequences from highly conserved repeats of a genome, particularly a combination of ERIC sequences and REP elements, are a convenient and accurate tool for the subspecies-specific discrimination and epidemiologic tracking of S. aureus.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.77-86
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• 2003

Pullorum disease due to Salmonella enterica subspecies enterica bioserovar Pullorum (S. pullorum) is reported to be an endemic disease in domestic poultry flocks. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of most of salmonella serotypes and other diverse bacterial species from animals and environmental samples in worldwide. Nowadays, PFGE has already been evaluated as a gold standards for molecular subtyping of salmonella serotypes compared with other molecular analysis methods. PFGE of XbaI digested chromosomal DNA from 23 strains of S. pullorum gave 5 distinctive pulsotypes (from SXPI to SXPV) with 5% confidence range of Dice coefficients, indicating that PFGE is very discriminative and that multiple clones of S. pullorum have been existed and diffused all of domestic poultry flocks industries since 1995. Two dominant pulsogroups (SXA & SXB) appeared as a major clones in this country, because they had consistently been recovered from diverse sources including both chicken organs and raw feed materials between 1995 and 1998. In addition, the matching percentage of PFGE profiles (PFP) among strains from both chickens and feed ingredients provides indirect evidence of the possible transmission of pullorum disease from contaminated raw feed ingredients for chicken production. In calculating of discrimination index (DI) for PFGE method by Simpson's index, DI was appeared as 0.917. Therefore, this index suggested that the present PFGE would seem to be a desirable and confident molecular typing method for S. pullorum strains. To our knowledge for pullorum disease, this is the first study to compare S. pullorum strains from chicken organs and feed samples using the PFGE.

• Korean journal of applied entomology
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• v.61 no.3
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• pp.399-408
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• 2022

There has been much debate on the morphometric divergence between the recently identified Apis cerana koreana and Apis cerana honey bees. The aim of this study was to obtain phenotypic information that can be used to compare A. c. koreana data with other A. cerana subspecies data from open resources and determine breeding results on the basis of morphometric traits. To differentiate A. c. koreana, we investigated 22 classic morphological characteristics; royal jelly secretion; and the weight of workers, queens, and drones of A. c. koreana bred in Korea. To define the selection results, we used the geometric morphometric method. The artificially selected A. c. koreana secreted significantly more royal jelly (1.18 times) than the naturally selected A. c. koreana, which positively influenced the health of the colonies. These honey bees were identified more clearly with the geometric morphometric method than with the classic morphometric method, which is traditionally used to determine the subspecies. Large trends were noted for A. c. koreana on the basis of our results and literature from the 1980s regarding A. cerana sizes in Korea (tarsal index, length of forewing, and cubital index were measured). The cluster analysis revealed the proximity of A. c. koreana, A. cerana in China, and A. c. indica on the basis of eight classic characters, which, perhaps, relay the origin of the honey bees. The results of this study defined the morphometric responses of A. c. koreana honey bees to geographic isolation, climate change, and selection, which are important to identify, protect, and preserve honey bee stock in Korea.

• Food Science of Animal Resources
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• v.28 no.3
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• pp.276-282
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• 2008

It is estimated that over 80% of deer antlers produced in the world are consumed in Korea. However, mislabeling or fraudulent replacement of costly antlers with cheaper ones is one of the most common problems in the domestic antler market. Therefore, there is a great need for the development of technology to identify species of antlers. This study was carried out to develop an accurate and reliable method for the identification and authentication of species or subspecies of antlers using DNA sequence analysis and comparison of mitochondrial cytochrome band D-loop region genes among antlers of five deer species, Cervus elaphus sibericus, Cervus elaphus canadensis, Cervus nippon, Cervus elaphus bactrianus and Rangifer tarandus. A variable region of cytochrome band D-loop genes was amplified using PCR with specifically designed primers and sequenced directly. The cytochrome band D-loop region genes showed different DNA sequences between the species of antlers and thus it is possible to differentiate between species on the basis of sequence variation. To distinguish between reindeer (Rangifer tarandus) antlers and other deer antlers, PCR amplicons of the cytochrome b gene were digested with the restriction enzymes NlaIV and TaqI, respectively, which generates a species-specific DNA profile of the reindeer. In addition, samples of 32 sliced antlers labeled Cervus elaphus sibericus from commercial markets were collected randomly and the mt DNA D-loop region of these antler samples was sequenced. Among the antler samples investigated, only 62.5% were from Cervus elaphus sibericus, and others were from Cervus elaphus bactrianus (25.0%), elk (Cervus elaphus canadensis) and reindeer (Rangifer tarandus). Our results suggest that DNA sequencing of mt DNA and PCR-RFLP methods using NlaIV and TaqI enzymes are useful for the identification and discrimination of deer antler species by routine analysis.