• The Journal of the Korean Society for Microbiology
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• v.35 no.5_6
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• pp.351-351
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• 2000

• Korean Journal of Veterinary Pathology
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• v.6 no.1
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• pp.1-5
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• 2002

A1celaphine herpesvirus 1 (AHV-1) is a causative agent of malignant catarrhal fever which is a fatal and a lymphoproliferative syndrome. AHV-1 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens or specific nucleic acids because of its low viral copies in the infected tissues. A new method, in situ PCR, is developed for the detection of AHV-1 nucleic acid in this study. Target sequences of AHV-1 open reading frame 50 gene were detected within AHV-1 infected MDBK cells. As compare with other molecular biological methods for the detection of AHV-1, in situ PCR was found to be more sensitive than in situ hybridization and to be less sensitive than nested PCR. However, nested PCR cannot afford to observe and differentiate AHV-1 infected cells. In situ PCR amplifies a target sequence within cells that can be visualized microscopically with increased sensitivity compared to detection by in situ hybridization. In situ PCR has wide applications for sensitive localization of low copy AHV-1 viral sequences within cells to investigate the role of viruses in a variety of clinical conditions and also provide the rapid, sensitive, and specific detection of AHV-1 infection.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• Fisheries and Aquatic Sciences
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• v.9 no.2
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• pp.70-74
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• 2006

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products. This sequencing strategy relies on 'nested' or 'tailed' primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. $5{\mu}L$) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.

• Biomedical Science Letters
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• v.8 no.3
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• pp.185-188
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• 2002

We have cloned the gene fur long QT syndrome in Korean genome and determined its detailed nucleotide sequence. Blood DNAs were isolated from 68 healthy individuals (including males and females) and the genomic DNAs were amplified by PCR method followed by automatic DNA sequencing. Entire sequence of the coding region for KCNEI was located in exon 3. PCR products were reexamined for the confirmation of KCNE1-specific amplification by nested PCR. KCNE1 mRNA was 436 bp. This corresponded to 129 amino acids. There was no recognizable difference between males and females. This study should contribute to the better understanding of long QT syndrome in Korean population.

• Parasites, Hosts and Diseases
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• v.51 no.2
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• pp.213-218
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• 2013

Malaria is a parasitic infection caused by Plasmodium species. Most of the imported malaria in Korea are due to Plasmodium vivax and Plasmodium falciparum, and Plasmodium ovale infections are very rare. Here, we report a case of a 24-year-old American woman who acquired P. ovale while staying in Ghana, West Africa for 5 months in 2010. The patient was diagnosed with P. ovale malaria based on a Wright-Giemsa stained peripheral blood smear, Plasmodium genus-specific real-time PCR, Plasmodium species-specific nested PCR, and sequencing targeting 18S rRNA gene. The strain identified had a very long incubation period of 19-24 months. Blood donors who have malaria with a very long incubation period could be a potential danger for propagating malaria. Therefore, we should identify imported P. ovale infections not only by morphological findings but also by molecular methods for preventing propagation and appropriate treatment.

• Parasites, Hosts and Diseases
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• v.52 no.6
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• pp.691-694
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• 2014

The purpose of this study was to conduct a survey of Dirofilaria immitis infection among stray cats in Korea using nested PCR. We included 235 stray cats (121 females and 114 males) and evaluated each for the presence of feline heartworm infection. Blood samples were collected from 135 cats in Daejeon, 50 cats in Seoul, and 50 cats from Gyeonggi-do (Province). Of the 235 DNA samples, 14 (6.0%) were positive for D. immitis. The prevalence of infection in male cats (8/114, 7.0%) tended to be higher than that in female cats (6/121, 5.0%), but the difference was not statistically significant. In each location, 8, 2, and 4 cats were positive for infection, respectively, based on DNA testing. No significant differences in the prevalence were observed among the geographic regions, although the rate of infection was higher in Gyeonggi-do (8.0%) than Daejeon (5.9%) and Seoul (4.0%). We submitted 7 of the 14 D. immitis DNA-positive samples for sequencing analysis. All samples corresponded to partial D. immitis cytochrome c oxidase subunit I gene sequences with 99% homology to the D. immitis sequence deposited in GenBank (accession no. FN391553). To the best of our knowledge, this is the first survey using nested PCR to analyze the prevalence of D. immitis in stray cats in Korea.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1453-1457
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• 2014

Background: The association of colorectal cancer with human cytomegalovirus (HCMV) is a controversial issue in cancer research. This study aimed to identify the HCMV virus in colorectal cancer tissues and to investigate the association of HCMV with colorectal cancer. In this study, 50 cancer tissue samples and 50 samples without colon cancer were studied in order to identify the HCMV virus through nested-polymerase chain reaction. The virus was identified in 15 cases of colorectal cancer tissues (15/50) and in 5 cases of normal tissues (5/50). Eight cases of adenocarcinoma tissues were in a moderately differentiated stage, and 7 cases had well-differentiated stage tissues that were positive for viral DNA. The findings were statistically evaluated at a significance level of p<0.05. The HCMV virus could playa role in creating malignancy and the progress of cancer through the process of oncomodulation.

• Research in Plant Disease
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• v.19 no.1
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• pp.36-44
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• 2013

Carnation is considered to be one of the top three cutting flowers in the world, which is a main crop with 21 billion annual volume of manufacture. The four carnation items such as cuttings, seed, plant and unrooted cuttings are imported and exported. Viruses can be easily transmitted during vegetative propagation of carnation. Carnation necrotic fleck virus (CNFV) and Carnation ringspot virus (CRSV) are designated as Korea plant quarantine viruses and inspected. This study was aimed to develop specific primer sets for easy and rapid detection of CNFV and CRSV. Two RT-PCR primer sets were efficiently amplified 288 and 447 bp fragments for CNFV and 503 549 bp fragments for CRSV. Furthermore, developed nested primer sets make possible to high sensitive detection and verification. CNFV nested PCR primer sets all produced band of 147 bp and CRSV nested PCR primer sets did bands of 395 and 347 bp. In addition, plasmid inserted 6 sequences in amplicon were used as a positive control to improve inspection confidence. The successful application of PCR module newly developed in this study will be highly useful for detect of CNFV and CRSV for quarantine inspections.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.3
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• pp.423-427
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• 2004

Seventy-six animals including cattle, sheep, horses, 6 species of zoo animals were employed for collection of fresh feces in order to detect verotoxigenic Esherichia coli (VTEC) by safe, quick and sensitive PCR-based molecular methods. Bacterial cell disruption with bead-beating followed by bacterial DNA purification with hydroxyapatide chromatography and gel filtration allowed DNA preparation from animal feces with high recovery and purity. A mountain goat was firstly shown by PCR and sequencing to shed verotoxin 2 gene (vt2) that was used to generate vt2 probe and second primer set for nested PCR to attempt more sensitive detection. Most sensitive nested PCR revealed that 45% of tested cattle and 47% of tested zoo animals were VTEC-positive, while least sensitive normal PCR detected VTEC from none of these animals except a mountain goat. Moderately sensitive detection by PCR in combination with hybridization suggested that the VTEC density varied between the VTEC-positive cattle.