• Parasites, Hosts and Diseases
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• v.48 no.4
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• pp.297-301
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• 2010

Recently, emerging waterbome protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from $10^1$ to $10^2$ oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium paNum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

• Journal of Veterinary Clinics
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• v.29 no.4
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• pp.306-308
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• 2012

A 1-year-old female pet rabbit (Lion head, BW: 1.1 kg) was present to the animal hospital for vaccination of viral hemorrhagic disease. This rabbit was adopted in 4-month-old, and contacted with wild cats in outdoors before adoption. We examined feces for Toxoplasma gondii by PCR analysis. We detected the presence of the 497 base pair fragment as a positive result. This is the first detection of T. gondii DNA in a pet rabbit from Korea by PCR analysis.

• Journal of Microbiology
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• v.38 no.1
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• pp.11-17
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• 2000

A new method was developed for the rapid analysis of diverse bacterial species in the natural environment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indigenous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.1 no.1
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• pp.83-89
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• 2020

Methods for detecting the presence of genetically modified (GM) crops are evolving to comply with legislation and to enhance monitoring by biotechnology companies and regulators. In order to cover a broad range of detection methods for a new GM crop, conventional multiplex PCR methods are required. Based on the genetic information on three GM alfalfa varieties (J101, J163, and KK179), which were recently approved in South Korea, we developed a fast, reliable, and highly specific multiplex polymerase chain reaction (PCR) method with basic PCR equipment and inexpensive reagents. To validate and verify the newly developed multiplex PCR method, we applied a limit of detection assay and random reference material analysis. We also monitored the unintentional environmental release of GM alfalfa in South Korea by performing the multiplex PCR analysis with 91 feral alfalfa specimens collected from 2000 to 2018. Our methodology is a sensitive, simple, quick, and inexpensive tool for detecting and identifying three GM alfalfa varieties.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.152-157
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• 2006

The purpose of this study was to evaluate the usefulness of the automated TB-PCR assay for the detection of Mycobacterium tuberculosis. The 807 cases were analyzed with their TB-PCR, AFB smear and culture in bronchial washing fluids, sputum and body fluids samples. The TB-PCR positive of the bronchial washing fluid, sputum and body fluids were 11.3%, 7.3% and 3.6%, respectively, in cases of AFB smear-negative and culture-negative. The sensitivity values of the bronchial washing fluid, sputum and body fluids were 93.3%, 100% and 50%, respectively, according to the culture result. The sensitivity of body fluids was lower than that of the bronchial washing fluid and sputum. The specificity values of the bronchial washing fluid, sputum and body fluids were 83.3%, 89.0% and 95.7%, respectively, according to the culture result. In conclusion, the automated TB-PCR assay proved to be a useful method for the detection of Mycobacterium tuberculosis in the bronchial washing fluid and sputum. But we think that there is still a need for us to study body fluids further.

• Parasites, Hosts and Diseases
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• v.51 no.1
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• pp.1-8
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• 2013

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

• Korean Journal of Plant Resources
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• v.21 no.3
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• pp.210-215
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• 2008

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer $Express^{TM}$ software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.

• Food Engineering Progress
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• v.21 no.2
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• pp.174-179
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• 2017

For preliminary molecular typing, PCR-based fingerprinting using random amplified polymorphic DNA (RAPD) is the method of choice. In this study, 14 bacterial strains were isolated from different Korean food sources, identified using 16S rRNA gene sequencing, and characterized through RAPD-PCR. Two PCR primers (239 and KAY3) generated a total of 130 RAPD bands, 14 distinct PCR profiles, 10 polymorphic bands, one monomorphic band, and four unique bands. Dendrogram-based analysis with primer 239 showed that all 14 strains could be divided into seven clades out of which clade VII had the maximum of seven. In contrast, dendrogram analysis with the primer KAY3 divided the 14 L. citreum strains into four clades out of which clade IV consisted of a maximum of 10 strains out of 14. This research identified and characterized bacterial populations associated with different Korean foods. The proposed RAPD-PCR method, based on sequence amplification, could easily identify and discriminate the lactic acid bacteria species at the strain-specific level and could be used as a highly reliable genomic fingerprinting tool.

• Journal of Korean Society of Forest Science
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• v.105 no.4
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• pp.422-428
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• 2016

Ginkgo (Ginkgo biloba L.) is one of the most appropriate roadside trees because of a good transplantation nature and ability to grow well in urban environment. Ginkgo is a dioecious species. Sex discrimination of ginkgo is possible through comparing morphological characters of reproductive organs. However, it needs more than about twenty years for reproductive organs to appear after sexual maturity. Until now, ginkgo trees for roadside plantation have been planted without discriminating the sex because ginkgo trees have been usually planted before sexual maturity. Ginkgo nuts from the female ginkgo trees planted along the roadside emit a foul odor, and make much pollution on the streets. Thus in this study a novel SCAR marker (SCAR-GBM) for the early sex discrimination was developed. Primers were developed on the basis of the sequence of male-specific RAPD variants reported previously. False-negative problem of SCAR marker, probably caused by dominant nature, was resolved by using multiplex PCR using primers of both the SCAR-GBM and a universal primer set of atp1 region in mitochondria DNA, which resulted in improved discrimination efficiency. The results showed that DNA bands of 1,039 bp were commonly amplified by the atp1 primer set in male and female trees, and SCAR-GBM markers of 675 bp were specifically amplified only in male trees. Reproducible and specific discrimination of the multiplex PCR was finally confirmed by applying multiple male and female individuals.