• Biomedical Science Letters
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• v.4 no.1
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• pp.43-56
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• 1998

A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

• Research in Plant Disease
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• v.17 no.2
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• pp.184-190
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• 2011

Ralstonia solanacearum, a causal agent of bacterium wilt is very difficult to control once the disease becomes endemic. Thus, Ralstonia solanacearum is a plant quarantine bacterium in many countries including Korea. In this study, we developed PCR assays, which can detect Ralstonia solanacearum from the Solanaceae seeds. Primers RS-JH-F and RS-JH-R amplified specifically a 401 bp fragment only from Ralstonia solanacearum race 1 and race 3. The nested PCR primers, RS-JH-F-ne and RS-JH-R-ne that were designed inside of 1st PCR amplicon amplified specifically a 131 bp fragment only from Ralstonia solanacearum race 1 and race 3. The primers did not amplify any non-specific DNA from the seed extracts of the Solanaceae including tomato and pepper. When detection sensitivity were compared using the Solanaceae seeds inoculated with target bacteria artificially, the nested PCR method developed in this study 100 times more sensitive than ELISA and selective medium. Therefore, we believe that the PCR assays developed in this work is very useful to detect Ralstonia solanacearum in the Solanaceae seeds.

• Food Engineering Progress
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• v.15 no.4
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• pp.370-375
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• 2011

In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monoytogenes, Staphyloocus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over $10^{5}$ CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over $10^{1}$ CFU/g, but only those of B. cereus were over $10^{3}$ CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

• Research in Plant Disease
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• v.23 no.4
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• pp.363-366
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• 2017

Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and Barley yellow dwarf virus (BYDV) have been identified as an important causative agents for an economically important disease of winter barley in Korea. In this study, a multiplex reverse transcription polymerase chain reaction (mRT-PCR) method was used for the simultaneous detection. Three sets of virus-specific primers targeted to the capsid protein coding genes of BaMMV, BaYMV and BYDV were used to amplify fragments that were 594 bp, 461 bp, and 290 bp, respectively. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by multiplex RT-PCR. The optimum primer concentrations and RT-PCR conditions were determined for the multiplex RT-PCR. The mRT-PCR assay was found to be a better and rapid virus diagnostic tool of specific barley diseases and potential for investigating the epidemiology of these viral diseases.

• Research in Plant Disease
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• v.16 no.2
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• pp.129-135
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• 2010

PCR primers were developed to detect Pseudomonas savastanoi pv. phaseolicola, a causal agent of halo blight that occurs in all species of common bean (Phaseolus vulgaris L.), from the bean seeds. A primer set, Psp-JHF and Psp-JH-R, specifically amplified 513 bp fragment from Pseudomonas savastanoi pv. phaseolicola only. A nested primer set, psp-JH-F-ne and psp-JH-R-ne, designed from the $1^{st}$ PCR amplicon, amplified 169 bp fragment. The primer sets did not amplify any non-specific DNA from the seed extracts of Fabaceae including 4 beans, 2 soybeans, and 2 peas. The detection sensitivity of the nested PCR method developed in this study was much higher than that of ELISA and selective medium. PCR assays developed in this study should be useful to detect Pseudomonas savastanoi pv. phasolicola from the bean seeds.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Research in Plant Disease
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• v.20 no.1
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• pp.21-24
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• 2014

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin is one of the most damaging diseases of Brassicaceae family. In this study, we developed species-specific primer sets for rapid and accurate detection of P. brassicae. The primer sets developed amplified a specific fragment only from P. brassicae DNA while they did not amplify a band from 10 other soilborne pathogens or from Kimchi cabbage. In sensitivity test, the species-specific primer set ITS1-1/ITS1-2 could work for approximately 10 spores/ml of genomic DNA showing more sensitivity and accuracy than previous methods. With quantitative real-time PCR test, the primer set detected less spores of P. brassicae than before, confirming that the species-specific primer set could be useful for rapid and accurate detection of P. brassicae.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.246-252
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• 2008

Tumor tissue is usually contaminated by normal tissue components, which reduces the sensitivity of analysis for exploring genetic alterations. Although microdissection has been adopted to minimize the contamination of tumor DNA with normal cell components, there is a concern over the amount of microdissected DNA not enough to be applied to array-CGH reaction. To amplify the extracted DNA, several whole genome amplification (WGA) methods have been developed, but objective comparison of the array-CGH outputs using different types of WGA methods is still scarce. In this study, we compared the performance of non-amplified microdissected DNA and DNA amplified in 2 WGA methods such as degenerative oligonucleotide primed (DOP)-PCR, and multiple strand displacement amplification (MDA) using Phi 29 DNA polymerase. Genomic DNA was also used to make a comparison. We applied those 4 DNAs to whole genome BAC array to compare the false positive detection rate (FPDR) and sensitivity in detecting copy number alterations under the same hybridization condition. As a result microdissected DNA method showed the lowest FPDR and the highest sensitivity. Among WGA methods, DOP-PCR amplified DNA showed better sensitivity but similar FPDR to MDA-amplified method. These results demonstrate the advantage and applicability of microdissection for array-CGH analysis, and provide useful information for choosing amplification methods to study copy number alterations, especially based on precancerous and microscopically invaded lesions.

• Korean Journal of Environmental Biology
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• v.19 no.4
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• pp.321-324
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• 2001

Groundwater samples were collected at 6 sites in Seoul area. DNA extraction from the sample was performed by the boiling method. Samples were boiled with guanidinium thyocyanate and phenol-chloroform. One set of primer was designed for amplification of 16S rDNA. For detection of denitrifying bacteria in groundwater sample, the author used primer sets consensus regions in gene sequences encoding the two forms of nitrite reductase (NIR), a key enzyme in the denitrification pathway. Two sets of PCR primer were designed to amplify $cd_1$-and Cu-nir. We confirmed the existence of denitrifying bacteria in 3 sites using $cd_1$-nir primer and in 4 sites using Cu-nir primer.

• The Plant Pathology Journal
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• v.20 no.4
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• pp.302-307
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• 2004

Three pairs of specific primers were developed for rapid and precise RT-PCR detection of three seed-transmissible viruses, namely Peanut clump virus (PCV, Pecluvirus), White clover mosaic virus (WCIMV, Potexvirus) and Carrot red leaf virus (CaRLV, Luteovirus). Each primer set was found in conserved region through multiple sequence alignment in the DNAMAN. Total nucleic acids extracted from PCV-, WCMV-, and CaRLV-infected seeds and healthy plants were used for RT-PCR detection using each virus-specific primer, Sizes of PCV, WCIMV, and CaRLV PCR products were 617bp (PCV-uni5 and PCV-uni3 primers), 561bp (WCMV-CP5 and WCMV-CP3 primers), and 626bp (CL1-UP and CL2-DN primers); which corresponded to the target sizes. Nucleotides sequences of each amplified cDNA were confirmed which belonged to the original virus. This study suggests that these virus-specific primer sets can specifically amplify viral sequences in infected seeds. Thus, they can be used for specific detection of three viruses (PCV, WCMV and CaRLV) from imported seed samples for plant quarantine service.