• Mycobiology
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• 제47권2호
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• pp.200-206
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• 2019

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker $7-2_{299}$ distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

• Journal of Microbiology
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• 제42권4호
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• pp.365-368
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• 2004

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA $\rightarrow$CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gin to Arg.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.490-495
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• 2007

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.

• Journal of Microbiology and Biotechnology
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• 제25권3호
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• pp.321-327
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• 2015

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64℃. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10³ CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.381-384
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• 2011

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial largesubunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

• Asian-Australasian Journal of Animal Sciences
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• 제21권1호
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• pp.124-130
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• 2008

Streptococcus (Strep.) agalactiae is one of the major pathogens of bovine mastitis and is the main cause of subclinical infection. This study attempted to develop a rapid PCR diagnosis procedure using milk samples collected on filter paper disks. Chromatographic filter paper was employed as the preservation media and kept at room temperature for one to four weeks. The revival rate of Strep. agalactiae kept on dried filter paper disks was affected by the pretreatment preservation time. The revival test suggested that not all the bacteria in artificially contaminated milk samples on the filter paper disks could be recovered. After that, a PCR based on the 16-23S intergenic spacer region of Strep agalactiae was performed. The results distinguished the strep. agalactiae from major pathogens of bovine mastitis at a $2{\times}10^2$ colony forming units (CFU)/ml level, which showed similar sensitivity to the results from liquid milk samples. The results also showed that milk samples collected on filter paper disks could be kept at room temperature for one to four weeks with little negative effect on sensitivity and specificity. The field test showed that the diagnostic sensitivity and specificity was 96.15% and 98.60%, respectively. In conclusion, the protocol will provide a rapid and economic procedure for the detection of bovine mastitis.

• BMB Reports
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• 제42권3호
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• pp.160-165
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• 2009

The temperate Staphylococcus aureus phage ${\Phi}11$ harbors cI and cro repressor genes similar to those of lambdoid phages. Using extremely pure ${\Phi}11$ Cro (the product of the ${\Phi}11$ cro gene) we demonstrated that this protein possesses a single domain structure, forms dimers in solution at micromolar concentrations and maintains a largely $\alpha$-helical structure even at $45^{\circ}C$. ${\Phi}11$ Cro was sensitive to thermolysin at temperatures ranging from $55-75^{\circ}C$ and began to aggregate at ${\sim}63^{\circ}C$, suggesting that the protein is moderately thermostable. Of the three homologous 15-bp operators (O1, O2, and O3) in the ${\Phi}11$ cI-cro intergenic region, ${\Phi}11$ Cro only binds efficiently to O3, which is located upstream of the cI gene. Our comparative analyses indicate that the DNA binding capacity, secondary structure and dimerization efficiency of thermostable ${\Phi}11$ Cro are distinct from those of P22 Cro and $\lambda$ Cro, the best characterized representatives of the two structurally different Cro families.

• Journal of Plant Biotechnology
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• 제44권3호
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• pp.235-242
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• 2017

Cudrania tricuspidata Bureau is a widely-used, medicinal, perennial and woody plant. Obtaining information about the genetic diversity of plant populations is highly important with regard toconservation and germplasm utilization. Although C. tricuspidata is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish Korean-specific ecotypes from other ecotypes from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the chloroplast and nuclear genomic sequences, which serve to to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the maturaseK (MatK) chloroplast intergenic region and nuclear ribosomal DNA internal transcribed spacer (ITS) regions. The SNP markers developed in this study are useful for rapidly identifying specific C. tricuspidata ecotypes from different regions.

• Journal of Microbiology
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• 제45권1호
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• pp.69-74
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• 2007

Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

• 한국동물위생학회지
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• 제34권3호
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• pp.201-208
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• 2011

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a pathogen of a variety of infections in horse. We studied biochemical and molecular characteristics of S. zooepidemicus isolated from the genital tract of Thoroughbred mares in Korea. Seventy-nine isolates were identified as S. zooepidemicus by biochemical and PCR method from 374 horses. The biochemical characteristics of S. zooepidemicus isolates were positive reaction of lactose and sorbitol. However, S. zooepidemicus isoltes were negative reaction of inulin, mannitol, raffinose, trehalose, aesculin hydrolysis, growth in 6.5% NaCl and variable reaction of maltose. Epidemiological investigations of S. zooepidemicus isolates were performed by fragment analysis of SzP (S. zooepidemicus protective protein) gene, CNE (collagen binding protein) gene and ISR (16s rRNA intergenic spacer region) gene using ABI Prism $3,130{\times}1$ Genetic Analyzer System. All isolates were shown single amplification size of 906 bp in CNE gene, but SzP and ISR gene were shown variable patterns of fragment size. The characteristics of S. zooepidemicus investigated in this study will be very useful for the prevention of infection and the studies of epidemiologic characteristics of S. zooepidemicus, causing the severe economic losses due to reproductive failures.