• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.77-83
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• 1998

돼지 생식기호흡기증후군(porcine reproductive and respiratory syndrome, PRRS) 바이러스가 웅돈에 감염되었을 경우에는 정충의 기형 등 정액의 질 저하와 더불어 정액에 바이러스가 함유되어 있어 종부시 모돈에 바이러스를 전파하는 것으로 알려져 있다. 감염된 웅돈의 정액으로 인공수정을 실시할 경우 농장의 모돈 전체에 순식간에 바이러스를 전파하여 막대한 피해를 유발할 가능성이 높다. 따라서 감염웅돈을 신속히 검색하여 격리함으로써 피해를 사전에 방지해야 하며, 이를 위해서 웅돈의 감염여부를 신속히 확진하는 진단법의 개발이 필요한 실정이다. PRRS의 진단을 위해서는 바이러스학적인 진단법으로는 바이러스 분리동정, 혈청학적인 방법으로 바이러스 분리동정이 필수적이나 검사시간이 많이 소요되고, 분리동정 자체가 까다로운 단점이 있다. 본 연구에서는 웅돈의 정액내에서 PRRSV 바이러스에 대한 유전자를 RT-PCR법으로 증폭하는 방법을 개발하였으며, 진단의 민감도를 높이기 위하여 Nested PCR법으로 재확인 할 경우, 바이러스의 역가가 $1TCID_{50}$만 함유되어 있어도 진단이 가능한 조건을 확립하였다. 이 방법을 이용할 경우 웅돈의 정액시료에 대한 PRRS 바이러스 감염여부를 신속, 정확하게 검색하여 감염웅돈을 통한 PRRS바이러스의 전파를 미리 차단할 수 있으므로 PRRS방제에 효과적으로 이용될 것으로 사료된다.

• 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.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.169-176
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• 1997

In this study, the feasibility of identification and genotypic differentiation of enteroviruses was investigated by using nested reverse transcription-polymerase chain reaction (nested RT-PCR), single-stranded conformation polymorphism (SSCP), and restriction fragment length polymorphism (RFLP) techniques. Two hundred seventy-four clinical samples were assayed by both nested RT-PCR and tube culture method using MRC-5 and MK cells; 58 (86.6%) out of 67 enterovirus culture-positive samples contained enteroviral RNA. In addition, 114 (55.1%) of 207 samples from patients with suspected enteroviral CNS disease with negative viral cultures were positive by the nested RT-PCR. The nested RT-PCR products were genotyped by the SSCP method and the results were compared with serotypes. We could differentiate 6 subtypes, 3 of which are similar to coxsackievirus B3, B5, echovirus 11, plus 3 other subtypes. RFLP cleaved with Sty I, Bgl I, and Xmn I yielded characteristic patterns for each laboratory strains. This study demonstrates the usefulness of the RT-PCR for the rapid diagnosis of enterovirus infection and the potentials of the SSCP method for differentiation of enterovirus strains.

• Korean Journal of Food Science and Technology
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• v.44 no.5
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• pp.638-642
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• 2012

Human pathogenic viruses such as hepatitis A and E virus (HAV and HEV), which lead to acute liver failure and death, are foodborne pathogens associated with the consumption of virus-contaminated meats, filter-feeding bivalves, fruits, and salads. Two of the three swine farms examined in this study had HAV and HEV positive stool samples in a nested RT-PCR assay. The use of the immunomagnetic separation (IMS) facilitated the separation of HAV through interactions between the ligand on the virion surface and the antibody from the swine feces containing both HAV and HEV. The nested RT-PCR analysis was performed for the detection of HAV obtained from hepatocarcinoma cell line (PLC/PRF/5) contaminated with eluent fraction of IMS. This indicated that IMS has the potential to simultaneously isolate and concentrate target viruses by changing antibodies linked on the magnetic beads.

• Biomedical Science Letters
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• v.25 no.4
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• pp.331-338
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• 2019

Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.147-155
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• 1998

Hantavirus is a genus of the Bunyaviridae family consisting following serotype groups: Hantaan, Seoul, Puumala, Prospect Hill, Thailand, Belgrade, Thotta palayam, Sin Nombre. Most of Hantavirus group have been associated with many clinically similar disease known collectively as hemorrhagic fever with renal syndrome (HFRS). Hantaan virus is the prototype of the genus hantavirus, originally isolated from Apodemus agrarius. Bat was found as a natural host for Hantaan virus in Lee's lab for the first time. Then, Hantaan-like virus was isolated Hantaan-like virus from bat. To identify hantaviruses that are present in Korea among bats, bats were collected from Jeong-Sun, Won-Joo, Chung-Ju and Hwa-Cheon area, RNA was isolated from lung and serum. RT-PCR was performed with a universal primer from M segment. Nested RT-PCR was carried out to differentiate Hantaan, Seoul and Puumala virus using serotype specific primers. As we expected, Hantaan viruses were detected in bats and Seoul virus was not detected. Interestingly, Puumala viruses were also detected in bats from Won-Ju, but not in other areas. Puumala virus is originally isolated from Clethrinomys glareolus, and cause light HFRS. Recently, Paradoxomis webbiana, a wild bird turn out to be a reservoir for Puumala virus in Korea. These data indicate that bat is a new natural reservoir of Puumala virus.

• Tuberculosis and Respiratory Diseases
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• v.72 no.2
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• pp.156-162
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• 2012

Background: The main goal of this study was to evaluate the diagnostic efficacy of reverse transcription-nested polymerase chain reaction (RT-nested PCR) in bronchial washing fluid with MAGE A1-6 common primers for the detection of lung cancers invisible by bronchoscopy. Methods: To determine the expression of MAGE A1-6 gene in 189 lung cancers diagnosed by conventional fluoroscopy-guided lung biopsy and 89 cancer-free controls, RT-nested PCR was performed in bronchial washing specimens. We analyzed MAGE A1-6 RT-nested PCR data according to tumor histology, stage, size, and compared them with cytological data. Results: 189 patients (111 cases in adenocarcinoma, 47 cases in squamous cell carcinoma, 22 cases in small cell lung carcinoma, and 9 cases in other cancers) and 89 benign patients were investigated. The expression of MAGE was performed by nested RT-PCR using common MAGE primer. Among 189 cancer patients, the expression rate of MAGE was 49.2%, and the positive predictive value was 89.4%. However, the expression rate of MAGE in patients with benign lesions was 12.4%. In peripheral lung cancer, the positive rate of MAGE expression was 57.4% in squamous cell carcinoma, 44.1% in adenocarcinoma and 59.1% in small cell lung cancer. Whereas the expression rate of bronchial washing cytology in peripheral lung cancer was 9.0% (p=0.011). Conclusion: MAGE RT-PCR in bronchial washing fluid gave us promising data for the detection of peripheral lung cancer. It could be a useful method for selecting diagnostic tools for peripheral lesions.

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.171-171
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• 2008

We monitored the occurrence of human enteric viruses in urban rivers by cell culture-PCR and RT-nested PCR. Water samples were collected monthly or semimonthly between May 2002 and March 2003 in four urban tributaries. Enteric viruses were detected by RT-nested PCR and cell culture-PCR based on a combination of Buffalo Green monkey kidney (BGMK) and A549 cell lines, followed by phylogenetic analysis of amplicons. By RT-nested PCR analysis, 45 (77.6%), 32 (55.2%), 32 (55.2%), 26 (44.8%), 12 (20.7%), 2 (3.4%), 4 (6.9%), and 4 (6.9%) of 58 samples showed positive results with adenoviruses, enteroviruses, noroviruses (NV) genogroup I (GI) and II (GII), reoviruses, hepatitis A viruses, rotaviruses and sapoviruses, respectively. Adenoviruses were most often detected and only eight (13.8%) samples were negative for adenoviruses and positive for other enteric viruses in the studied sites. Thirty-one (77.5%) of the 40 samples were positive for infectious adenoviruses and/or enteroviruses based on cell culture-PCR, and the frequency of positive samples grown on A549 and BGMK (65.0%) was higher than that grown on BGMK alone (47.5%). The occurrence of each enteric virus, except reoviruses and hepatitis A viruses was not statistically correlated with the water temperature and levels of fecal coliforms according to Binary logistic regression model. By sequence analysis, most strains of adenoviruses and enteroviruses detected in this study are similar to the causative agent of viral diseases in Korea and most NV GI- and GII-grouped strains were closely related to the reference strains from China and Japan, and GII/4-related strains had similar sequences to strains recognized as a worldwide epidemic outbreak. Our results suggested that monitoring human enteric viruses is necessary to improve microbial quality and cell culture-PCR using the combination of A549 and BGMK cells and the adenovirus detection by PCR could be useful for monitoring viral contamination in the aquatic environment.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.190-198
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• 2021

Human Aichivirus (Aichivirus A; AiV-A) is a positive-sense single-strand RNA non-enveloped virus that has been detected worldwide in various water environments including sewage, river, surface, and ground over the past decade. To develop a method with excellent sensitivity and specificity for AiV-A diagnosis from water environments such as groundwater, a combination capable of reverse transcription (RT)-nested polymerase chain reaction (PCR) was developed based on existing reported and newly designed primers. A selective method was applied to evaluate domestic drinking groundwater samples. Thus, a procedure was devised to select and subsequently identify RT-nested PCR primer sets that can successfully detect and identify AiV-A from groundwater samples. The findings will contribute to developing a better monitoring system to detect AiV-A contamination in water environments such as groundwater.

• The Plant Pathology Journal
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• v.30 no.2
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• pp.178-182
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• 2014

Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea.