• Plant Biotechnology Reports
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• v.4 no.4
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• pp.293-301
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• 2010

The feasibility of two strategies for transgene pyramiding using Agrobacterium-mediated transformation was investigated to develop a transgenic potato (Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) (Phthorimaea operculella (Zeller)). In the first approach, cry1Ac9 and cry9Aa2 genes were introduced simultaneously using a kanamycin (nptII) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry1Ac9 gene, using a hygromycin resistance (hpt) selectable marker gene, into an existing line transgenic for a cry9Aa2 gene and a kanamycin resistance (nptII) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.4
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• pp.163-168
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• 2013

Objective: Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. Results: Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. Conclusion: This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases.

• Animal Bioscience
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• v.35 no.8
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• pp.1121-1128
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• 2022

Objective: This study aimed to identify the genetic diversity and population structure of Mongolian horse populations according to the province of residence (Khentii, KTP; Uvs, USP; Omnogovi and Dundgovi, GOP; Khovsgol, KGP) using 14 microsatellite (MS) markers. Methods: A total of 269 whole blood samples were obtained from the four populations (KTP, USP, GOP, KGP) geographically distinct provinces. Multiplex polymerase chain reaction (PCR) was conducted using 14 MS markers (AHT4, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, and VHL20), as recommended by the International Society for Animal Genetics. Capillary electrophoresis was conducted using the amplified PCR products, alleles were determined. Alleles were used for statistical analysis of genetic variability, Nei's DA genetic distance, principal coordinate analysis (PCoA), factorial corresponding analysis (FCA), and population structure. Results: On average, the number of alleles, expected heterozygosity (H_Exp), observed heterozygosity (H_Obs), and polymorphic information content among all populations were 11.43, 0.772, 0.757, and 0.737, respectively. In the PCoA and FCA, GOP, and KGP were genetically distinct from other populations, and the KTP and USP showed a close relationship. The two clusters identified using Nei's DA genetic distance analysis and population structure highlighted the presence of structurally clear genetic separation. Conclusion: Overall, the results of this study suggest that genetic diversity between KTP and USP was low, and that between GOP and KGP was high. It is thought that these results will help in the effective preservation and improvement of Mongolian horses through genetic diversity analysis and phylogenetic relationships.

• Journal of Veterinary Science
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• v.24 no.6
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• pp.82.1-82.12
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• 2023

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [bla_TEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

• Pediatric Infection and Vaccine
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• v.26 no.2
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• pp.99-111
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• 2019

Purpose: Respiratory syncytial virus (RSV) and human rhinovirus (hRV) are the most common causes of child respiratory viral infections. We aimed to investigate epidemiological and clinical characteristics of RSV and hRV single infections and coinfections. Methods: Nasopharyngeal aspirates of hospitalized children aged <5 years were tested using multiplex reverse transcription polymerase chain reaction (RT-PCR) from October 2014 to April 2017. Their medical records were retrospectively reviewed. Results: RSV or hRV was detected in 384 patients who divided into 3 groups: patients with RSV (R group, n=258); patients with hRV (H group, n=99); and patients with both (RH group, n=27). The R group (median age, 6 months) consisted of 248 (96.1%) patients with lower respiratory tract infection (LRTI), and 14 (5.4%) needed oxygen inhalation. Infants aged <12 months (63.2%) had respiratory difficulty and were supplied oxygen more often. The H group (median age, 16 months) consisted of 56 (56.6%) patients with LRTI, 4 (4%) required oxygen inhalation, and 1 (1.0%) required mechanical ventilation. Infants (40.4%) showed longer hospitalization compared to patients aged ${\geq}12$ months (5 vs. 4 days, P<0.05). The RH group consisted of 24 (88.9%) patients with LRTI, and 2 (7.4%) needed oxygen inhalation. Hospitalization days and oxygen inhalation and mechanical ventilation rates did not differ between single infections (R and H groups) and coinfections (RH group). Conclusions: RSV was detected more often in younger patients and showed higher LRTI rates compared to hRV. Single infections and coinfections of RSV and hRV showed no difference in severity.

• Pediatric Infection and Vaccine
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• v.14 no.1
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• pp.69-74
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• 2007

Purpose : Human coronanviruses (hCovs) including hCoV-229E and hCoV-OC43 have been known as etiologic agents of the common colds and were regarded as clinically insignificant agents. However, recent identification of hCoV-NL63 and hCoV-HKU1 in children with lower respiratory tract infections has evoked the clinical concerns about their prevalence and the clinical significance of these hCoVs in children. This study was performed to investigate the prevalence of hCoVs in children with community-acquired pneumonia. Methods : From March 2006 to January 2007, nasopharyngeal specimens collected from children hospitalized with pneumonia, were tested for the presence of common respiratory viruses (respiratory syncytial virus, influenza A, influenza B, parainfluenza viruses, and adenovirus) using multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Human metapneumovirus (hMPV) infection was excluded by nested RT-PCR using primers for the F-gene. To detect the different strains of hCoVs, nested RT-PCR assays specific for hCoVNL63, hCoV-OC43, hCoV-229E, and hCoV-HKU1 were performed. Results : Out of the 217 nasopharyngeal aspirate from children aged under 15 years, respiratory syncytial virus (RSV) was detected in 32 patients, hMPV in 18, human parainfluenza virus in 10, influenza virus A in 2, and adenovirus in 6. HCoVs were detected by RT-PCR in 8 (3.7%) of the 217 patients, hCoV-229E in 1, hCoV-NL63 in 3, and hCoVOC43 in 4 patients. HCoV-HKU1 was not detected in this study population. Conclusion : Recently identified hCoV-NL63 and hCoV-HKU1 seemed to have a little clinical significance in Korean children with severe or hospitalized community-acquired pneumonia.

• Research in Plant Disease
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• v.12 no.2
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• pp.139-143
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• 2006

Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.4
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• pp.401-406
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• 2017

In this study, using the VITEK 2 system, 74 samples (22.6%) out of 327 specimens were identified by the growth of Enterococcosel media (EV6 agar) supplemented with $6{\mu}g/mL$ of vancomycin. Enterococcus faecium was identified as 55 strains (74.3%), Enterococcus casseliflavus as 2 strains (2.7%), Enterococcus avium as 1 strain (1.4%), and Enterococcus gallinarum as 16 strains (21.6%). Among the 55 phenotypes of Enterococcus faecium, 42 (76.4%), 9 (16.4%), and 4 strains (7.3%) showed the vanA, vanB, and vanC phenotype, respectively. The 16 strains of Enterococcus gallinarum and 2 strains of Enterococcus casseliflavus showed the vanC phenotype and the 1 strain of Enterococcus avium had the vanB phenotype. The one strain of Enterococcus faecium propagated only in EV4 and was susceptible to both vancomycin and teicoplanin according to the antimicrobial susceptibility test using the VITEK 2 system. The vancomycin resistance phenotype gene was not detected by PCR. A total of 327 specimens were cultured in Enterococcosel broth supplemented with $6{\mu}g/mL$ of vancomycin (EV6 broth), and 120 strains (36.7%) were isolated. These 120 strains were subjected to vancomycin resistant genotyping by a multiplex real-time polymerase chain reaction and 51 strains (42.5%) showed vanA; 5 strains (4.2%) showed vanA and vanC; and 18 strains (15%) showed vanC. Vancomycin resistance genotypes were not detected in the remaining 46 strains (38.3%).

• Pediatric Infection and Vaccine
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• v.23 no.2
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• pp.87-93
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• 2016

Purpose: An outbreak of influenza virus is uncommon in neonatal intensive care unit (NICU). The clinical presentation of influenza virus infection in neonates is diverse. This study was aimed to report an outbreak of influenza A in a NICU and to investigate the clinical characteristics of influenza virus infection in neonates especially preterm infants during the 2011-2012 influenza season in Korea. Methods: We reviewed the medical records of 29 patients who were evaluated by respiratory virus multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) at NICU of Kosin University Gospel Hospital during the 2011-2012 seasonal influenza outbreak in Korea. Results: Eleven patients (37.9%) were influenza A virus RT-PCR positive during the survey periods. They were all preterm infants and three of them had no symptoms. Eight patients had symptoms and it was fever (18%, 2/11), respiratory difficulty (72.7%, 8/11) without symptoms of upper respiratory infection, and gastrointestinal symptoms (27.3%, 3/11). The median duration of symptom was 5 days. There were differences of duration of admission at the test of respiratory RT-PCR, Clinical Risk Index for Babies (CRIB) score, use of mechanical ventilation, and use of dexamethasone before infection between influenza A virus RT-PCR positive and negative group. All 11 patients with influenza A were discharged without any complications. Conclusions: The symptoms of influenza A virus infection in the preterm infants is nonspecific. Influenza A virus should be considered as a possible cause of infection in NICU during the influenza season in the community.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.384-391
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• 2009

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.