• Tuberculosis and Respiratory Diseases
• /
• v.71 no.4
• /
• pp.249-253
• /
• 2011

Background: Mycobacterial infection is a problem throughout the world along with the increase of immunocompromised patients. For this reason, there have been many methods for faster and more accurate diagnosis. In this study, we evaluated several laboratory methods for mycobacterial infection. Methods: From January to December 2009, 635 specimens were cultured with mycobacteria growth indicator tube (MGIT) and Ogawa media. Polymerase chain reaction (PCR) was performed with the AdvanSure tuberculosis (TB)/non-tuberculosis mycobacterium (NTM) real-time PCR Kit (LG Life Sciences, Seoul, Korea). The 69 samples showing positive culture results were identified with the AdvanSure Mycobacteria Genotyping Chip Kit (LG Life Science, Seoul, Korea). Results: Sixty-nine (10.9%) out of 635 samples showed positive results for mycobacterial culture. Among the 635 samples, 64 were positive in MGIT, but only 42 were positive in Ogawa media. Of the 635 samples, 607 (95.6%) showed the same results between MGIT and Ogawa and the results of 579 (95.4%) were also consistent with the TB/NTM real-time PCR results. However, in the case of NTM, only one (1/24, 4.2%) was positive in PCR. In the Mycobacteria genotyping chip analysis, the most frequently identified NTM species in descending order were M. avium, M. intracellulare, M. chelonae and M. abscessus. Conclusion: Culturing with a combination of MGIT and Ogawa is recommended to increase the recovery rate of mycobacteria. Although PCR missed a reasonable number of NTM, it is faster and usually gives results that concur with those from the culture. The appropriate combination of diagnostic methods with clinical correlation are necessary.

• Journal of Veterinary Clinics
• /
• v.32 no.2
• /
• pp.154-157
• /
• 2015

A major cause of diarrhea in a dog is an infection with bacteria which include Salmonella spp., Campylobacter (C.) spp., and Clostridium (Cl.) spp.. It is fastidious to identify these bacteria by the culture. The purpose of this experiment is to devise the method for detecting Cl. perfringens, C. jejuni, C. coli, and Salmonella spp. with rapid and high sensitivity. The fecal samples collected from 71 normal and 66 diarrheic dog feces were used to compare the prevalence of the enteric pathogens and to develop a multiplex real-time polymerase chain reaction (PCR) assay for clinical use. Detection of Cl. perfringens, C. coli, and C. jejuni in diarrhea feces was higher than normal feces. A developed multiplex real-time PCR is useful for determining the presence and quantity of pathogen-specific or other unique sequences with in a fecal sample.

• Korean Journal of Veterinary Service
• /
• v.41 no.2
• /
• pp.111-118
• /
• 2018

This study was carried out to investigate the prevalence of honeybee (Apis mellifera) diseases in Gwangju area. From November 2016 to August 2017, 89 samples were collected from 33 apiculture farms and reverse transcriptase-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), and real time PCR were conducted. 14 infectious pathogens, including seven viruses, two bacteria, three fungi, and two parasites, were investigated from random apiculture farms in Gwangju. The percentage of infectious pathogens were as follows: Stonebrood (76.4%), Deformed wing virus (51.7%), Nosema (27.0%) in PCR and RT-PCR. This result indicated that Stonebrood was most prevalent disease in Gwangju area. And we could get similar results from real time PCR. 84.8% of farms have more than two of infectious pathogens. Stonebrood and Deformed wing virus were major diseases in almost all seasons and Black queen cell virus disease was especially prevalent in May.

• Journal of Chest Surgery
• /
• v.44 no.2
• /
• pp.123-130
• /
• 2011

Background: The aim of the present study was to identify chromosomal loci that contribute to the pathogenesis of aortic dissection (AD) in a Korean population using array comparative genomic hybridization (CGH) and to confirm the results using real-time polymerase chain reaction (PCR). Materials and Methods: Eighteen patients with ADs were enrolled in this study. Genomic DNA was extracted from individual blood samples, and array CGH analyses were performed. Four corresponding genes with obvious genomic changes were analyzed using real-time PCR in order to assess the level of genomic imbalance identified by array CGH. Results: Genomic gains were most frequently detected at 8q24.3 (56%), followed by regions 7q35, 11q12.2, and 15q25.2 (50%). Genomic losses were most frequently observed at 4q35.2 (56%). Real-time PCR confirmed the results of the array CGH studies of the COL6A2, DGCR14, PCSK6, and SDHA genes. Conclusion: This is the first study to identify candidate regions by array CGH in patients with ADs. The identification of genes that may predispose an individual to AD may lead to a better understanding of the mechanism of AD formation. Further multicenter studies comparing cohorts of patients of different ethnicities are warranted.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.7
• /
• pp.993-1003
• /
• 2013

Methanotrophs are the most important sink of $CH_4$, which is a more highly potent greenhouse gas than $CO_2$. Methanotrophic abundance and community diversity in cover soils from two typical semi-aerobic landfills (SALs) in China were detected using real-time polymerase chain reaction (real-time-PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA genes, respectively. Real time-PCR showed that Type I methanotrophs ranged from $1.07{\times}10^6$ to $2.34{\times}10^7$ copies/g soil and that of Type II methanotrophs from $1.51{\times}10^7$ to $1.83{\times}10^8$ copies/g soil. The ratio of Type II to Type I methanotrophic copy numbers ranged from 5.61 to 21.89, indicating that Type II methanotrophs dominated in SAL. DGGE revealed that Type I methanotrophs responded more sensitively to the environment, changing as the community structure varied with different soil types and locations. Methylobacter, Methylosarcina, and Methylomicrobium for Type I, and Methylocystis for Type II were most prevalent in the SAL cover layer. Abundant interflow $O_2$ with high $CH_4$ concentration in SALs is the reason for the higher population density of methanotrophs and the higher enrichment of Type II methanotrophs compared with anaerobic landfills and other ecosystems, which proved a conclusion that increasing the oxygen supply in a landfill cover layer would greatly improve $CH_4$ mitigation.

• Journal of Ginseng Research
• /
• v.41 no.3
• /
• pp.326-329
• /
• 2017

Background: Cultivated ginseng is often introduced as a substitute and adulterant of Russian wild ginseng due to its lower cost or misidentification caused by similarity in appearance with wild ginseng. The aim of this study is to develop a simple and reliable method to differentiate Russian wild ginseng from cultivated ginseng. Methods: The mitochondrial NADH dehydrogenase subunit 7 (nad7) intron 3 regions of Russian wild ginseng and Chinese cultivated ginseng were analyzed. Based on the multiple sequence alignment result, a specific primer for Russian wild ginseng was designed by introducing additional mismatch and allele-specific polymerase chain reaction (PCR) was performed for identification of wild ginseng. Real-time allele-specific PCR with endpoint analysis was used for validation of the developed Russian wild ginseng single nucleotide polymorphism (SNP) marker. Results: An SNP site specific to Russian wild ginseng was exploited by multiple alignments of mitochondrial nad7 intron 3 regions of different ginseng samples. With the SNP-based specific primer, Russian wild ginseng was successfully discriminated from Chinese and Korean cultivated ginseng samples by allele-specific PCR. The reliability and specificity of the SNP marker was validated by checking 20 individuals of Russian wild ginseng samples with real-time allele-specific PCR assay. Conclusion: An effective DNA method for molecular discrimination of Russian wild ginseng from Chinese and Korean cultivated ginseng was developed. The established real-time allele-specific PCR was simple and reliable, and the present method should be a crucial complement of chemical analysis for authentication of Russian wild ginseng.

• Journal of Korean Society of Water and Wastewater
• /
• v.23 no.2
• /
• pp.199-205
• /
• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

• Journal of Ginseng Research
• /
• v.43 no.3
• /
• pp.482-487
• /
• 2019

Background: The mixed-cultivation of different Panax ginseng cultivars can cause adverse effects on stability of yield and quality. K-1 is a superior cultivar with good root shape and stronger disease resistance. DNA markers mined from functional genes are clearly desirable for K-1, as they may associate with major traits and can be used for marker-assisted selection to maintain the high quality of Korean ginseng. Methods: Five genes encoding pathogenesis-related (PR) proteins of P. ginseng were amplified and compared for polymorphism mining. Primary, secondary, and tertiary structures of PR5 protein were analyzed by ExPASy-ProtParam, PSSpred, and I-TASSER methods, respectively. A coding single nucleotide polymorphism (SNP)-based specific primer was designed for K-1 by introducing a destabilizing mismatch within the 3' end. Allele-specific polymerase chain reaction (PCR) and real-time allele-specific PCR assays were conducted for molecular discrimination of K-1 from other cultivars and landraces. Results: A coding SNP leading to the modification of amino acid residue from aspartic acid to asparagine was exploited in PR5 gene of K-1 cultivar. Bioinformatics analysis showed that the modification of amino acid residue changed the secondary and tertiary structures of the PR5 protein. Primer KSR was designed for specific discrimination of K-1 from other ginseng cultivars and landraces. The developed real-time allele-specific PCR assay enabled easier automation and accurate genotyping of K-1 from a large number of ginseng samples. Conclusion: The SNP marker and the developed real-time allele-specific PCR assay will be useful not only for marker-assisted selection of K-1 cultivar but also for quality control in breeding and seed programs of P. ginseng.

• Korean Journal of Veterinary Service
• /
• v.45 no.2
• /
• pp.87-99
• /
• 2022

A novel porcine circovirus 4 (PCV4) was recently emerged in Chinese and Korean pig herds, which provided epidemiological situation where three pathogenic PCVs, PCV2, PCV3, and newly emerged PCV4, could co-infect pig herds in these countries. In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) method was developed for the rapid and differential detection of these viruses. The assay specifically amplified each viral capsid gene, whereas no other porcine pathogenic genes were detected. The detection limit of the assay was below 10 copies/µL and the assay showed high repeatability and reproducibility. In the clinical evaluation using 1476 clinical samples from 198 Korean pig farms, the detection rates of PCV2, PCV3 and PCV4 by the tqPCR assay were 13.8%, 25.4%, and 3.8%, respectively, which were 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4, with a κ value (95% CI) of 1 (1.00~1.00). The prevalence of PCV2, PCV3, and PCV4 at the farm levels were 46.5%, 63.6%, and 19.7%, respectively. The co-infection analysis for tested pig farms showed that single infection rates for PCV2, PCV3, and PCV4 were 28.8%, 44.4%, and 9.6%, respectively, the dual infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 12.6%, 3.5%, and 5.1%, respectively, and the triple infection rate for PCV2, PCV3, and PCV4 was 1.5%. These results demonstrate that three pathogenic PCVs are widely spread, and their co-infections are common in Korean pig herds, and the newly developed tqPCR assay will be useful for etiological and epidemiological studies of these pathogenic PCVs.

• Biomedical Science Letters
• /
• v.26 no.4
• /
• pp.302-309
• /
• 2020

Human Coxsackievirus B5 (HuCoxV-B5) infection has been associated with various diseases such as myocarditis, aseptic meningitis, hand-foot-and mouth-disease, and insulin-dependent diabetes. HuCoxV-B5 is a virus transmitted through the fecal-oral route and is detected in clinics, aquatic environments, food, shellfish, etc. and is one of the more important viruses in public health because of its incidence rate reported worldwide. In this study, a combination of SYBR Green-based real-time PCR primers for molecular diagnosis including monitoring of HuCoxV-B5 was selected and the optimal reaction conditions were established. Compared with the previously reported TaqMan probe-based real-time PCR method, assessments including a sample applicability test were performed. Results showed that the real-time PCR method developed in this study was suitable for a molecular diagnostic technique for detecting HuCoxV-B5. This study is expected to contribute to efforts in responding to safety accidents in public health because the proposed method facilitates rapid diagnosis of clinical patients. It can also be used as a specific monitoring tool of HuCoxV-B5 in non-clinical areas such as aquatic environments among others.