• Asian Pacific Journal of Cancer Prevention
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• v.16 no.7
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• pp.3003-3007
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• 2015

Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC had markedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant difference in free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detection was obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2006.11a
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• pp.88-89
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• 2006

Fowl cholera is an infectious disease caused by .Pasteurella multocida, affecting domesticated and wild birds. It usually appears as a septicemia of sudden onset with high morbidity and mortality, but chronic conditions that characterized by localized infections often occur. 13wks broiler breeders were submitted to the Kyung-pook national university for diagnosis. Clinical signs included approximately 1% mortality, severe lameness, ruffled feathers and swollen and/or cloudy eyes. At necropsy, the outstanding lesions were seen swollen hock joint, which were suppurative or caseous exudates, inflammation of conjunctiva, severe pneumonia and epicarditis. The causative agent was isolated from the hock joint, liver, sinus and sternum of the chickens, and performed physiological and biochemical test. To identify the serotype of P. multocida, capsular serotyping was conducted by multiplex polymerase chain reaction (PCR). In the antimicrobial susceptibility test, the isolates were resistance to the aminoglycosides. In this study, we confirmed chronic fowl cholera (FC) caused by P. multocida in broiler breeders in Korea.

• 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.29 no.1
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• pp.94-99
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• 2023

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

• Journal of Ginseng Research
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• v.41 no.1
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• pp.31-35
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• 2017

Background: Korean ginseng (Panax ginseng) is a well-known medicinal plant of Oriental medicine that is still in practice today. Until now, a total of 11 Korean ginseng cultivars with unique features to Korean ginseng have been developed based on the pure-line-selection method. Among them, a new cultivar namely G-1 with different agricultural traits related to yield and content of ginsenosides, was developed in 2012. Methods: The aim of this study was to distinguish the new ginseng cultivar G-1 by identifying the unique single-nucleotide polymorphism (SNP) at its 45S ribosomal DNA and Panax quinquefolius region than other Korean ginseng cultivars using multiplex amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR). Results: A SNP at position of 45S ribosomal DNA region between G-1, P. quinquefolius, and the other Korean ginseng cultivars was identified. By designing modified allele-specific primers based on this site, we could specifically identified G-1 and P. quinquefolius via multiplex PCR. The unique primer for the SNP yielded an amplicon of size 449 bp in G-1 cultivar and P. quinquefolius. This study presents an effective method for the genetic identification of the G-1 cultivar and P. quinquefolius. Conclusion: The results from our study shows that this SNP-based approach to identify the G-1 cultivar will be a good way to distinguish accurately the G-1 cultivar and P. quinquefolius from other Korean ginseng cultivars using a SNP at 45S ribosomal DNA region.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.14 no.3
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• pp.1191-1196
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• 2013

Among Gram-negative pathogens in Korea, the incidence of resistance to third generation cephalosporins is becoming an ever-increasing problem. The production of extended-spectrum ${\beta}$-lactamase (ESBL) is the main mechanism of bacterial resistance to a third-generation cephalosporins and monobactams. Accurate identification of the ESBL genes are necessary for surveillance and epidemiological studies of the mode of transmission in the hospital. This study was conducted to detect the genes encoding ESBL of 46 K. penumoniae isolated from Daejeon, Chungnam and Chungbuk regional university hospitals from February to August in 2012. The phenotypes of the isolated specimens were examined according to the combination disc test (CDT) by the Clinical and Laboratory Standards Institute (CLSI). Forty two ESBL producing K. penumoniae isolates could be detected using ceftazidime (CAZ) discs with and without clavulanate (CLA). By CDT, 42 K. pneumoniae strains were confirmed to be ESBL strains. Genotyping was performed by multiplex PCR with type-specific primers. By PCR analysis, TEM gene in 46 strains, SHV gene in 37 strains and CTX-M genes in 14 strains were identified. Ten isolates did carry genes encoding ESBLs of all types TEM, SHV and CTX-M. The multiplex polymerase chain reaction (PCR) analysis was better to detect and differentiate ESBL producing K. penumoniae strains in clinical isolates.

• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2003.10a
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• pp.27-27
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• 2003

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections are considered difficult to distinguish clinically and histopathologically. Prompt differentiation between PEDV- and TGEV-associated enteritis would greatly facilitate the management of disease in countries where PEDV and TGEV are epizootic. Rapid differential diagnosis and treatment are crucial to reducing mortality and morbidity from PEDV- and TGEV-induced enteritis in piglets. The objective for this study was to develop a protocol to differentiate between PEDV and TGEV directly from formalin-fixed, paraffin-embedded tissue, using a multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. (omitted)

• Journal of Microbiology
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• v.44 no.1
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• pp.92-97
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• 2006

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to $0.05\;pM/{\mu}l$. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.7
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• pp.916-920
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• 2013

The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.90-96
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• 2015

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.