• Research in Plant Disease
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• v.10 no.1
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• pp.53-57
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• 2004

Cucumber green mottle mosaic virus (CGMMV) was a major pathogen of watermelon and had affected seriously to watermelon production in Korea. Rapid and sensitive detection method of CGMMV associated with bottle gourd (Lagenafia siceraria) seeds was developed by using RT-PCR in this study. A pair of primeri Wmfl and Wmrl, specific for CGMMV was designed from coat protein gene sequences of CGMMV-W and used for amplifying 420 bp product in RT-PCR. To simplify the virus extraction procedure and reduce an inhibitor from the extract for the RT-PCR, some methods using ethanol precipitation, double filtration, polyethylene glycol precipitation and phenol/chloroform/isoamyl alcohol extraction procedure were compared and the phenol/chloroform/isoamyl alcohol extraction procedure was selected by its enhanced sensitivity. This detection method using the selected extraction step and the primers for RT-PCR could reliably detect an infected level of one CGMMV-infested seed in 1,000 seeds. This rapid and sensitive RT-PCR assay provides auseful tool for the specific detection of CGMMV in bottle gourd seed samples containing high levels of back-ground inhibitors.

• Development and Reproduction
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• v.18 no.3
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• pp.167-172
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• 2014

In the present study, muscle tissues were obtained separately from individuals from Atlantic hairtail population (AHP), Gunsan hairtail population (GHP) and Chinese hairtail population (CHP), respectively. The seven decamer primers were used to generate the shared loci, specific, unique shared loci to each population and shared loci by the three hairtail populations. Here, averagely, a decamer primer generated 64.7 amplified products per primer in the AHP population, 55.7 in GHP population and 56.4 in CHP population. The number of unique shared loci to each population and number of shared loci by the three populations generated by genetic analysis using 7 decamer primers in AHP, GHP and CHP population. 119 unique shared loci to each population, with an average of 17 per primer, were observed in the AHP population, and 28 loci, with an average of 4 per primer, were observed in the CHP population. The hierarchical dendrogram point out three main branches: cluster 1 (ATLANTIC 01 ~ ATLANTIC 07), cluster 2 (GUNSAN 08 ~ GUNSAN 14) and cluster 3 (CHINESE 15 ~ CHINESE 21). The shortest genetic distance displaying significant molecular difference was between individuals' CHINESE no. 16 and CHINESE no. 18 (0.045). In the long run, individual no. 01 of the AHP population was most distantly related to CHINESE no. 19 (genetic distance = 0.430). Consequently, PCR analysis generated on the genetic data displayed that the geographic AHP population was widely separated from CHP population, while individuals of CHP population were fairly closely related to those of GHP population.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1281-1287
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• 2009

The distribution pattern of the phylum Acidobacteria, a previously uncultured bacterial group, was investigated by molecular ecological analyses of global soil samples collected from pristine ecosystems across five continents. Acidobacterial 16S rDNAs were observed in almost all soil samples, and members of acidobacterial primer group A were detected in all samples that harbored the phylum Acidobacteria. Other primer groups, Y, G, and O, showed limited distribution patterns. We further divided the primer groups into acidobacterial subdivisions (class-level). Subdivisional distribution patterns were determined by comparing the observed T-RFs with theoretical T-RFs predicted by in silico digestion of acidobacterial 16S rDNAs. Consistent with the PCR results obtained with subgroup-specific primers, T-RFLP analyses showed that acidobacterial subdivision 1 belonging to primer group A was present in the majority of the soil samples. This study revealed that the phylum Acidobacteria could be globally distributed. At the subdivisional level, acidobacterial subdivision 1 might be the most widely distributed group in this phylum, indicating that members of subdivision 1 might be adapted to various soil environments, and members belonging to other subdivisions might be restricted to certain geographic regions or habitats.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.91-98
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• 2016

The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at $55^{\circ}C$ and 25 cycles at $60^{\circ}C$) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at $60^{\circ}C$) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.

• Research in Plant Disease
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• v.17 no.3
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• pp.376-380
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• 2011

The bacterial speck of tomato caused by Pseudomonas syringae pv. tomato leads to serious economic losses especially on fruits of susceptible genotype. Thus, Pseudomonas syringae pv. tomato is a plant quarantine bacterium in many countries including Korea. In this study, we developed specific PCR assays for detection of the bacterium from tomato seeds. A specific primer set is designed from the hrpZ gene for specific detection of Pseudomonas syringae pv. tomato. A 501 bp PCR product corresponding to hrpZ gene was amplified only form Pseudomonas syringae pv. tomato strains, but no PCR product was amplified from other tomato bacterial pathogens, such as Pseudomonas syringae pv. glycinea, P. syringae pv. maculicola, P. syringae pv. atropurpurea, P. syringae pv. morsprunorum, and from other P. syringae pathovar strains. The nested-PCR primer set corresponding to an internal fragment of the 501 bp sequence (hrpZ) gine was used to specific detection of Pseudomonas syringae pv. tomato in tomato seed. A 119 bp PCR product using nested PCR primer was highly specific and sensitive to detect low level of Pseudomonas syrigae pv. tomato in tomato seeds. We believe that the PCR assays developed in this study is very useful to detect Pseudomonas syringae pv. tomato from the tomato seeds.

• International Journal of Oral Biology
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• v.37 no.3
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• pp.109-114
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• 2012

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In this study, we screened for differentially expressed genes (DEGs) in periodontitis compared with normal tissue using an annealing control primer (ACP) system. By ACP RT-PCR analysis, we obtained about 160 amplicons, 8 of which were found to be differentially expressed. DEGs in patients with periodontitis were thus successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in the screen may also enhance our understanding of the pathogenesis of periodontitis.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1113-1117
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• 2012

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Biomedical Science Letters
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• v.10 no.4
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• pp.333-339
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• 2004

A multiplex PCR assay targeting the yst and 16S rRNA genes of Yersinia enterocolitica was developed to specifically identify pathogenic Y. enterocolitica from pure culture. Simultaneous amplification of 145 and 416 bp fragments of the yst and 16S rRNA genes of Y. enterocolitica was obtained using the primer pairs in a single reaction. Validation of the assay was performed with the reference Yersinia strains and other members of the family Enterobacteriaceae. The defined primer pairs amplified the targeted sequence from only pathogenic Y. enterocolitica strains, whereas none of the other bacterial species yielded any amplified fragments. Within an assay time of 4 h, this assay offers a very specific, reliable, and inexpensive alternative to the conventional phenotypic assays used in clinical laboratories to identify pathogenic Y. enterocolitica.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.46 no.4
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• pp.328-333
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• 2001

Allium is one of the largest genera, which has more than 700 species. PCR by URP (universal rice primer) primers was carried out to get phylogenetic information on 26 species, 62 accessions of subgenus Rhizirideum. The accessions were divided into seven groups at 0.76 similarity level. A. tuberosum (Chinese chives) and A. ramosum represented high similarity of 0.91. A. montanum, A. nutans, A. senescens, A. libani, A. odorum, A. austrosibiricum, and A. narcissiflorium grouped at 0.80 similarity. Some of the wild species, such as A. prostratum, A. polyrhizum, A. odorum, and A. mongolicum, showed different band patterns according to polyploidy, occurrence of B-chromosome, collection site, and origin.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2000.11a
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• pp.59-75
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• 2000

This study was carried out to develop a DNA marker for identifying between Korean cattle (Hanwoo) and other breeds. First experiment was performed to isolate Hanwoo specific DNA marker at sequence characterized amplified regions (SCARs). Five breeds of cattle including Hanwoo, Holstein, Hereford, Angus and Charolais were represented with the from 8 to 20 individuals. Fourteen primers of 300 arbitrary primers of 10 nucleotides showed reproducible polymorphism across the breeds. An amplified band of 0.9 kb in the primer MG-3 showed the specificity to Holstein breed. And MG-6 and MG-12 detected the Hereford and Hanwoo specific markers at the size of 2.0 kb and 1.0 kb, respectively. A 1.0 kb band of MG-12 was cloned and sequenced. A SCAR primer was designed based on the obtained sequences. It was possible to identify the Hanwoo from Holstein breed. Second experiment was carried out to observe the genotype frequencies of MC1R in 1,044 samples of imported beef and eight different cattle breeds including Hanwoo, Holstein, Angus, Brown-Swiss, Charolais, Limousin, Simmental and Hereford. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no.Y19103). A size of 350 bp was amplified by polymerase chain reaction(PCR), digested with two different restriction enzyme, BsrFI and MspA II, and electrophoresed in 2.5% Metaphore agarose gel for determination of genotypes. Genotype frequencies of Hanwoo were 0.10 in E+e and 0.90 in ee. Allele ED was shown in all of Holstein and Angus breeds tested which have black coat color phenotypes. We suggested that SCAR marker and the bovine MC1R gene could be used as a DNA marker for distinguishing beef between Hanwoo and Holstein.