• The Plant Pathology Journal
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• 제34권6호
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• pp.490-498
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• 2018

Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent cross-reactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and $3.86{\times}10^2cells$ for B. glumae and $5.85{\times}10^2cells$ for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as $1.86{\times}10^3cells$ per rice seed for B. glumae and $1.04{\times}10^4cells$ per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.

• The Plant Pathology Journal
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• 제19권3호
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• The Plant Pathology Journal
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• 제29권3호
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• pp.338-343
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• 2013

A detection system based on a multiplex reverse transcription (RT) polymerase chain reaction (PCR) was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV), lily mottle virus (LMoV), lily symptomless virus (LSV). Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single- or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV) by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.280-283
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• 1998

We have cloned the RNA polymerase sigma-like factors from a wide range of actinomycetes by using specific primers with the polymerase chain reaction (PCR). The specific oligonucleotide primers were designed on the basis of amino acid sequences of conserved regions from HrdA, B, D of Streptomyces griseus as well as from the rpoD box of many eubacteria. The consensus sequences were from the rpoD box and helix-turn-helix motif involved in -35 recognition. The designed primers were successfully applied to amplify the DNA fragments of the hrd homolog genes from 8 different strains of actinomycetes which produce a wide variety of important antibiotics. The 480 bp of the DNA fragment was amplified from all 8 strains, and it was identified as a part of hrdA and hrdB as we designed. The deduced amino acid sequence of PCR-amplified DNA fragments were highly homologous to those of other known RNA polymerase sigma factors of S. griseus and Streptomyces aureofaciens. Therefore, this study with specifically designed primers will support rapid cloning of the RNA polymerase sigma factors which recognize different classes of promoters from actinomycetes, and it will also be helpful in understanding the relationship of promoters and sigma factors leading to heterogeneity of RNA polymerases in actinomycetes.

• 동의생리병리학회지
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• 제18권3호
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• pp.825-829
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• 2004

This study was carried out to differentiate the origins of Astragalus membranaceus Bunge and A. membranaceus Bunge var. mogholicus Nakai. To identify the variation of the RAPD patterns between domestic and foreign Astragalus species, 40 random primers were applied to ten accessions of A. membranaceus and six accessions of A. membranaceus var. mogholicus genomic DNA, respectively, Ten primers of 40 primers could be used to discriminate the origins and 33 polymorph isms among 44 scored DNA fragments (33 fragments are specific for A. membranaceus and A. membranaceus var. mogholicus) were generated using these primers, 75.0 % of which were polymorphic. Especially, three primers of ten primers, OPA17, OPA11 and OPB11, were useful to differentiate between domestic and foreign Astragalus species. RAPD data from the 10 primers were used for cluster analysis and cluster analysis of RAPD markers showed that the two groups are distinct genetically. Consequently, RAPD analysis was a useful method to discriminate between A. membranaceus and A. membranaceus var. mogholicus.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.282-285
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• 2004

The polyene antibiotics including nystatin, pimaricin, amphotericin and candicidin are a family of most promising antifungal polyketide compounds, typically produced by rare actinomycetes species. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly homologous biosynthetic genes among polyene-producers such as polyketide synthase (PKS) and cytochrome P450 hydroxylase (CYP) genes. Based on amino acid sequence alignment among actinomycetes CYP genes, the highly-conserved regions specific for only polyene CYP genes were identified and chosen for degenerate PCR primers, followed by the PCR-screening with various actinomycetes genomic DNAs. Among tested several polyene non-producing actinomycetes strains, Pseudonorcardia autotrophica strain was selected based on the presence of PCR product with polyene-specific CYP gene primers, and then confirmed to contain a cryptic novel polyene hydroxylase gene in the chromosome. These results suggest that the polyene-specific hydroxylase gene PCR should be an efficient way of screening and isolating potentially-valuable cryptic polyene antibiotic biosynthetic genes from various microorganisms including rare actinomycetes.

• Parasites, Hosts and Diseases
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• 제56권3호
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• pp.295-300
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• 2018

Human sparganosis was diagnosed by morphological and genetic analyses in Korea. The complete mitochondrial genomes of Spirometra erinaceieuropaei and S. decipiens isolated in Korea have been recorded. Present study was performed to provide information to diagnose the etiologic agent of sparganosis by multiplex PCR using mitochondrial genome sequences of S. erinaceieuropaei and S. decipiens. In an effort to examine the differential diagnosis of spirometrid tapeworms, multiplex PCR assays were performed on plerocercoid larvae of S. erinaceieuropaei and S. decipiens. The PCR products obtained using species-specific primers were positively detected in all PCR assays on mixture of S. erinaceieuropaei and S. decipiens DNA. S. erinaceieuropaei-specific bands (239 bp and 401 bp) were obtained from all PCR assays using a mixture of S. erinaceieuropaei-specific primers (Se/Sd-1800F and Se-2018R; Se/Sd-7955F and Se-8356R) and S. erinaceieuropaei template DNA. S. decipiens-specific bands (540 bp and 644 bp) were also detected in all PCR assays containing mixtures of S. decipiens-specific primers (Se/Sd-1800F and Sd-2317R; Se/Sd-7955F and Sd-8567R) and S. decipiens template DNA. Sequence analyses on these species-specific bands revealed 100% sequence identity with homologous regions of the mtDNA sequences of S. erinaceieuropaei and S. decipiens. The multiplex PCR assay was useful for differential diagnosis of human sparganosis by detecting different sizes in species-specific bands.

• The Plant Pathology Journal
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• 제32권2호
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• pp.162-167
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• 2016

Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit, can be divided into three biovars (biovars 1, 2, and 3). Strains belonging to biovar 1 produce phaseolotoxin and were isolated in Japan and Italy before 2008. Strains of biovar 2 produce coronatine instead of phaseolotoxin and have been isolated only in Korea. Strains belonging to biovar 3 produce neither phaseolotoxin nor coronatine and are responsible for the global outbreak of bacterial canker of kiwifruit in recent years. The biovar 3-specific primer set was developed in a previous work. In this study, two sets of PCR primers specific to strains of biovars 1 and 2, respectively, were developed based on random amplified polymorphic DNA analyses. Primers PsaJ-F and PsaJ-R produced a 481-bp region with genomic DNA of biovar 1 strains, whereas primers PsaK-F and PsaK-R amplified a 413-bp region present only in the genome of biovar 2 strains.

• Food Science and Biotechnology
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• 제16권1호
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• pp.67-70
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• 2007

Mackerel (Scomber japonicus) often causes severe allergic reactions in sensitive people. Food containing undeclared mackerel may pose a risk to such people. The major allergenic protein in fish such as mackerel, codfish, and Alaska pollack has been found to be parvalbumin. In this study, we developed a polymerase chain reaction (PCR) method to detect mackerel DNA using primers corresponding to the parvalbumin gene. We cloned and sequenced 1.5 kb of parvalbumin gene by PCR using mackerel genomic DNA as a template. Nucleotide sequence analysis of genomic parvalbumin gene, composed of 4 exons and 3 introns, allowed the selection of two pairs of oligonucleotide primers specific for mackerel. These primers successfully enabled PCR amplification of specific regions of genomic parvalbumin DNA from mackerel, but no amplification from 8 other fish samples, surimi, and 6 boiled fish pastes. The sensitivity of this method was sufficient to detect 5 ng of purified mackerel DNA mixed with 50 ng of surimi DNA. This rapid and specific method for the detection of allergenic mackerel would be beneficial in reducing food allergy caused by the ingestion of hidden allergen in processed food.

• 한국패류학회지
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• 제29권3호
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• pp.163-169
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• 2013

In the present study, the seven primers BION-33, BION-34, BION-37, BION-41, BION-44, BION-45 and BION-42 generated the total number of loci, average number of loci per lane and specific loci in Hongseong, Yeosu and Goheung population of F. mutica, respectively. 7 primers generated 19 specific loci in the Hongseong population, 29.3 in the Yeosu population and 23.1 in the Goheung population, respectively. Especially, the decamer primer BION-37 generated 7 unique loci to each population, which were identifying each population, approximately 700 bp in Hongseong population. In this study, the dendrogram obtained by the seven primers indicates three genetic clusters: cluster 1 (HONGSEONG 01-HONGSEONG 07), cluster 2 (YEOSU 08-YEOSU 14) and cluster 3 (GOHEUNG 15-GOHEUNG 21). Among the twenty one cockles, the shortest genetic distance that displayed significant molecular differences was between individuals 17 and 19 from the Goheung population (genetic distance = 0.051), while the longest genetic distance among the twenty-one cockle individuals that displayed significant molecular differences was between individuals HONGSEONG no. 03 and YEOSU no. 12 (genetic distance = 0.616). Relatively, individuals of YEOSU population were fairly closely related to that of GOHEUNG population. Ultimately, PCR fragments revealed of in this study may be useful as a DNA marker the three geographic populations to distinguish.