• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.90-95
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• 1999

A species-specific 760 base pair(bp) BamHI to EcoRI DNA fragment(fMG-2) of lipoprotein gene was isolated from a Mycoplasma gallisepticum(M gallisepticum) genomic library. Based on the DNA sequence data of fMG-2, a pair of 25bp primers was synthesized. When used in the polymerase chain reaction(PCR), 732bp DNA products were amplified from 6 standard strains and 10 field isolates of M gallisepticum, but not from 2 Mycoplasma synoviae and 7 other Mycoplasma species. The lower detection limit was 100fg of the genomic DNA. Identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with AseI, DraI, EcoRV and SspI.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• Parasites, Hosts and Diseases
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• v.45 no.1 s.141
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• pp.69-74
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• 2007

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction(PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each(520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.103-112
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• 2003

A multiplex PCR assay, which allows simultaneous detection of vancomycin resistant genotypes and Enterococcus species-specific genes, was developed. Vancomycin resistant enterococci (VRE) from chickens and humans could be detected for vanA, vanB, vanC-1, vanC-2, $ddl_{E.faecium}$ and $ddl_{E.faecalis}$ by multiplex PCR. Eight isolates of VRE from humans (n=11) had $ddl_{E.faecium}$ and vanA, and 3 isolates of the VRE had $ddl_{E.faecium}$ and vanB. One isolate of VRE from chickens (n=6) had $ddl_{E.faecium}$ and vanA, and 5 isolates of the VRE had only vanA. E. faecium, E. faecalis, E. gallinarum and E. casseliflavus were also confirmed for the species-specific gene by multiplex PCR. This multiplex PCR could detect E. faecium, E. faecalis, E. gallinarum, E. casseliflavus, vanA, vanB, vanC-1 and vanC-2, simultaneously. The PCR assay established in the present study can be an alternative to time-consuming biochemical tests and antibiotic susceptibility tests of Enterococcus spp.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1492-1495
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• 2008

Successful control of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, requires a specific and reliable diagnostic tool. A pathovar-specific PCR assay was developed for the rapid and accurate detection ofthe plant pathogenic bacterium Xanthomonas oryzae pv. oryzicola in diseased plant. Based on differences in a membrane fusion protein gene of Xanthomonas oryzae pv. oryzicola and other microorganisms, which was generated from NCBI (http://www.ncbi.nlm.nih.gov/) and CMR (http://cmr.tigr.org/) BLAST searches, one pair of pathovar-specific primers, XOCMF/XOCMR, was synthesized. Primers XOCMF and XOCMR from a membrane fusion protein gene were used to amplity a 488-bp DNA fragment. The PCR product was only produced from 4 isolates of Xanthomonas oryzae pv. oryzicola among 37 isolates of other pathovars and species of Xanthomonas, Pectobacterium, Pseudomonas, Burkholderia, Escherichia coli, and Fusarium oxysporum f.sp. dianthi. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.131-140
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• 2020

Solanum stoloniferum, one of the wild tetraploid Solanum species belonging to the Solanaceae family, is an excellent resource for potato breeding owing to its resistance to several important pathogens. However, the sexual hybridization of S. stoloniferum with S. tuberosum (potato) is hampered due to the sexual incompatibility between the two species. To overcome this and introgress the various novel traits of S. stoloniferum in cultivated potatoes, cell fusion can be performed. The identification of the fusion products is crucial and can be achieved with the aid of molecular markers. In this study, the chloroplast genome sequence of S. stoloniferum was obtained by next-generation sequencing technology, and compared with that of six other Solanum species to identify S. stoloniferum-specific molecular markers. The length of the complete chloroplast genome of S. stoloniferum was found to be 155,567 bp. The structural organization of the chloroplast genome of S. stoloniferum was similar to that of the six other Solanum species studied. Phylogenetic analysis of S. stoloniferum with nine other Solanaceae family members revealed that S. stoloniferum was most closely related to S. berthaultii. Additional comparison of the complete chloroplast genome sequence of S. stoloniferum with that of five Solanum species revealed the presence of six InDels and 39 SNPs specific to S. stoloniferum. Based on these InDels and SNPs, four PCR-based markers were developed to differentiate S. stoloniferum from other Solanum species. These markers will facilitate the selection of fusion products and accelerate potato breeding using S. stoloniferum.

• Journal of Plant Biotechnology
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• v.47 no.2
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• pp.141-149
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• 2020

Solanum hougasii, one of the wild Solanum species, has been widely used in potato breeding since it exhibits excellent resistance to diverse important pathogens. S. hougasii can be directly crossed with the cultivated tetraploid potato (S. tuberosum) owing to its EBN (Endosperm Balanced Number) value of 4, which is same as that of S. tuberosum although it is an allohexaploid. In this study, the complete chloroplast genome sequence of S. hougasii was obtained by next-generation sequencing technology, and compared with that of the chloroplast genome of seven other Solanum species to identify S. hougasii-specific PCR markers. The length of the complete chloroplast genome of S. hougasii was 155,549 bp. The structural organization of the chloroplast genome in S. hougasii was found to be similar to that of seven other Solanum species studied. Phylogenetic analysis of S. hougasii with ten other Solanaceae family members revealed that S. hougasii was most closely related to S. stoloniferum, followed by S. berthaultii, and S. tuberosum. Additional comparison of the chloroplast genome sequence with that of five other Solanum species revealed five InDels and 43 SNPs specific to S. hougasii. Based on these SNPs, four PCR-based markers were developed for the differentiation of S. hougasii from other Solanum species. The results obtained in this study will aid in exploring the evolutionary and breeding aspects of Solanum species.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• Journal of Microbiology
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• v.38 no.2
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• pp.105-108
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• 2000

Oligonucleotide primers amplifying a 344 bp fragment on the integrin-like protein alpha-INT1p gene (${\alpha}$INT1) of Candida albicans were synthesized for screenign of C. albicans from clinicalsamples by the polymerase chain reaction (PCR). The PCR specifically amplified DNA from C. albicans and none from any other Candida, fungal, or human DNA in standard used here. The PCR assay showed that the primers (LH1 and LH2) were specific for 26 isolates of C. albicans from clinical smaples, whereas the positive fragment, 344 bp, was not amplified from 15 clinical isolates including 14 other medically important Candida species and an isolate of Saccharomyces cerevisiae. PCR was conducted on the urine samples of 20 patients and 4 samples were C. albicans positive. The detection limit of the PCR assay for C. albicans was shown to be approximately 10 cells/ml saline. The PCR system using 344 bp ${\alpha}$INT1 as a target is more specific and rapid than the conventional culture method, and the sensitive detection method is applicable to clinical diagnosis of C. albicans infections.

• Journal of Food Hygiene and Safety
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• v.37 no.1
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• pp.15-20
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• 2022

The purpose of this study was to compare the conventional culture method, enzyme immune method and the PCR method using species-specific primer in the analysis on the Salmonella spp. found in domestically distributed pet foods. For the study, Salmonella spp. were detected from 175 samples. From the conventional culture method and the PCR method, two samples (jerky and corn gluten) were determined as positive. Also, from the enzyme immune method, one sample (corn gluten) was test-positive. The study revealed that application of the PCR method with species-specific primer allows better distinguishment between the species of the strain collected from the samples than the conventional culture method and/or the enzyme immune method.