• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.130-135
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• 2004

The marine dinoflagellate genus Alexandrium, which produces PSP toxins, has a global distribution. As human-assisted dispersal of the species has been suggested, it is important to develop molecular tools to trace the dispersal pathway. To screen population-specific DNA sequences that differentiate Korean and Japanese A. catenella, we targeted the area downstream of the chloroplast psbA gene using PCR with population-specific DNA primers followed by RFLP (restriction fragment length polymorphism) analysis and sequencing. The RFLP patterns of the PCR products divided Korean and Japanese A. catenella regional isolates into three types: Korean, Japanese, and type CMC3, isolated from Korea. We sequenced the PCR products, but found no similar gene in a homology search. The molecular phylogeny inferred from the sequences separated the Korean and Japanese A. catenella strains, as did the RFLP patterns. However, the Japanese isolates included two slightly different sequences (types J and K), while the Korean sequence was the same as the Japanese K type. In addition, a unique sequence was found in the Korean strains CMC2 and CMC3. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers designed from the type J sequence yielded PCR products for Japanese strains only, showing that the unknown gene can be used for a population analysis of Korean and Japanese A. catenella.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1467-1470
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• 2012

We compared rapid fingerprinting using repetitive sequencebased PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1001-1010
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• 1997

Background : PCR technique is useful in diagnosis of pulmonary tuberculosis. But, its sensitivity and specificity is some different among several studies. Our aim is compare our PCR results with other's previous PCR results in AFB smear negative patients. Methods : PCR were performed in patients that their disease were suspected as active pulmonary tuberculosis and that their initial serial sputum AFB smear results were negative. Total number of patients studied by PCR technique was 177. Also, we analyzed the data only in patients whose bronchial washing fluid AFB smear was negative. And the primer had been used was IS 6110. Results : In our retrograde study, the number of patients who are diagnosed as having active pulmonary tuberculosis, inactive pulmonary tuberculosis and nontuberculous pulmonary disease was 99, 28, 50, respectively. In the sputum study, the sensitivity of PCR is 41.5% (27 PCR positive cases/65 active TBc cases). And the sensitivity of TB culture is 53.8% (35 TB culture positive cases/65 active TBc cases). In the bronchial washing specimen study, the sensitivity of PCR is 53.8% (21 PCR positive cases/39 active TBc cases). And the sensitivity of TB culture is 43.6% (17 TB culture Positive cases/39 active TBc cases). The specificity of PCR in our study is 94.9%. (74 PCR negative cases/78 inactive TBc or nontubereulous cases) In the cases of patients who were never takened anti-TBc medication, the sensitivity of PCR (45.6%--25 positive cases/55 cases) is some lower than culture (58.2%--32 positive cases/55 cases). In the cases of patients who had been takened anti-TBC medication. the sensitivity of PCR (60%--18 positive cases/30 cases) is some superior than culture (50%--15 positive cases/30 cases). Conclusion : We think that PCR results in cases of sputum AFB smear negative patients is nearly same as culture. And PCR is especially useful in patients who had been takened anti-TBc medication on admission.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.87-91
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• 2011

The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Journal of Life Science
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• v.16 no.5
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• pp.729-733
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• 2006

Cryptosporidium is a waterborne pathogenic parasite which causes diarrhea. Immunomagnetic separation-immunofluorescent assay (IMS-IFA) has been a widely adopted for Cryptosporidium detection as standard method. However, this method does not provide information about viability or infectivity of Cryptosporidium. Therefore, many researchers have studied viability or infectivity analyses of Cryptosporidium with various methods such as vital staining, in vitro excystation, RT-PCR, cell culture, and mouse infection assay. In this study, two direct RT-PCR methods, cell culture RT-PCR and cell culture IFA were compared for sensitivity and other characteristics. The results showed that direct RT-PCR method with HSP70 genes had the highest sensitivity with detection up to 1 viable cell of Cryptosporidium. The infectious Cryptosporidium were detected up to 10 to 25 cells by cell culture methods in combination with RT-PCR and IFA. The infectious Cryptosporidium were apt to be quantified by cell culture IFA.

• Food Science of Animal Resources
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• v.34 no.5
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• pp.717-723
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• 2014

A rapid and specific PCR assay for the simultaneous detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in foods was developed to reduce the detection time and to increase sensitivity. Multiplex PCR developed in this study produced only actA, fliC, hbl, invA, ileS amplicons, but did not produce any non-specific amplicon. The primer sets successfully amplified the target genes in the multiplex PCR without any non-specific or additional bands on the other strains. The multiplex PCR assays also amplified some target genes from five pathogens, and multiplex amplification was obtained from as little as 1 pg of DNA. According to the results from the sensitivity evaluation, the multiplex PCR developed in this study detected 10 cells/mL of the pathogens inoculated in milk samples, respectively. The results suggested that multiplex PCR was an effective assay demonstrating high specificity for the simultaneous detection of five target pathogens in food system.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.231-237
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• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

• Food Science of Animal Resources
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• v.40 no.4
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• pp.628-635
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• 2020

The objective of this study was to detect three non-halal meat products consisted of dog, pork, and rat species in meatball using novel multiplex-PCR with 12S rRNA gene as target sites. A total of 33 self-made meatballs were used, and they were grouped into eleven types of meatball based on meat species origin contained in the meatballs. Each type consisted of three meatballs. Extraction of genomic DNA from the meatballs was used as a DNA template for simplex-, duplex-, and multiplex-PCR processes. The result of simplex-PCR, duplex-PCR, and multiplex-PCR showed that the 12S rRNA primer gene successfully amplified DNA for each species bovine, dog, pig, and rat, which are respectively indicated by 155, 244, 357, and 491 bp of DNA bands. In addition, multiplex-PCR with 12S rRNA gene primers can be uniquely and accurately used for detection bovine, dog, pig, and rat species on beef meatball in one reaction.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.