• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.105-109
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• 2015

Andean potato latent virus (APLV) is a phytopathogenic virus that belongs to the Group IV (+) sense ssRNA viruses of the genus Tymovirus. It mainly infects potatoes and is specified as a controlled quarantine virus in Korea. In this study, two primer sets of RT-PCR and nested PCR [set 2 ($404{\rightarrow}259bp$) and set 23 ($501{\rightarrow}349bp$)], were selected, which can rapidly and accurately diagnose APLV in quarantine sites. In addition, a modified-positive control plasmid is development, can possible verification of laboratory contamination in diagnosis of APLV detection. The PCR-base system developed in this study is expected to diagnose APLV and contribute to the plant quarantine in Korea.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• 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.

• Korean Journal of Food Science and Technology
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• v.37 no.6
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• pp.1018-1023
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• 2005

Simplified procedure was developed for concentrating and detecting poliovirus and norovirus in oysters. Viruses were seeded into oyster tissue homogenates and concentrated through polyethylene glycol (PEG) precipitation, chloroform or Freon extraction, with additional PEG precipitation. Amount of viruses was evaluated using poliovirus plaque assay. Virus recovery during concentration procedure was approximately 16.4-26.0%. For defection, viral RNAs in oysters were examined using one-step RT-PCR after extraction with Trizol. Dilution or capturing of viral RNA using silica gel membrane allowed viruses to be detected by RT-PCR, whereas viruses could not be removed using $QIAshredder^{TM}$ Homogenizer, which is effective in removing RT-PCR inhibitors in lettuce and hamburgers. Freon extraction, generally used to concentrate viruses found in food, could be substituted with chloroform extraction using this procedure; no difference could be observed between detection limits of whole oyster extracts and digestive organ extracts indicating that RT-PCR inhibitors were distributed evenly throughout whole tissues. Nested PCR greatly improved efficiency of this procedure. Overall, this procedure could remove sufficient amount of inhibitors to allow detection of norovirus in oysters.

• Pediatric Infection and Vaccine
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• v.14 no.1
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• pp.69-74
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• 2007

Purpose : Human coronanviruses (hCovs) including hCoV-229E and hCoV-OC43 have been known as etiologic agents of the common colds and were regarded as clinically insignificant agents. However, recent identification of hCoV-NL63 and hCoV-HKU1 in children with lower respiratory tract infections has evoked the clinical concerns about their prevalence and the clinical significance of these hCoVs in children. This study was performed to investigate the prevalence of hCoVs in children with community-acquired pneumonia. Methods : From March 2006 to January 2007, nasopharyngeal specimens collected from children hospitalized with pneumonia, were tested for the presence of common respiratory viruses (respiratory syncytial virus, influenza A, influenza B, parainfluenza viruses, and adenovirus) using multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Human metapneumovirus (hMPV) infection was excluded by nested RT-PCR using primers for the F-gene. To detect the different strains of hCoVs, nested RT-PCR assays specific for hCoVNL63, hCoV-OC43, hCoV-229E, and hCoV-HKU1 were performed. Results : Out of the 217 nasopharyngeal aspirate from children aged under 15 years, respiratory syncytial virus (RSV) was detected in 32 patients, hMPV in 18, human parainfluenza virus in 10, influenza virus A in 2, and adenovirus in 6. HCoVs were detected by RT-PCR in 8 (3.7%) of the 217 patients, hCoV-229E in 1, hCoV-NL63 in 3, and hCoVOC43 in 4 patients. HCoV-HKU1 was not detected in this study population. Conclusion : Recently identified hCoV-NL63 and hCoV-HKU1 seemed to have a little clinical significance in Korean children with severe or hospitalized community-acquired pneumonia.

• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2003.10a
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• pp.26-26
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• 2003

Hepatitis E virus (HEV) has been recognized as a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries. The taxonomy of HEV is not clear and the virus remains unclassified. The objective of this study was to optimize conditions and procedures to detect swine HEV in formalin-fixed, paraffin-embedded tissues by nested RT-PCR and compare this detection method with in situ hybridization. (omitted)

• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2003.10a
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• pp.27-27
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• 2003

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections are considered difficult to distinguish clinically and histopathologically. Prompt differentiation between PEDV- and TGEV-associated enteritis would greatly facilitate the management of disease in countries where PEDV and TGEV are epizootic. Rapid differential diagnosis and treatment are crucial to reducing mortality and morbidity from PEDV- and TGEV-induced enteritis in piglets. The objective for this study was to develop a protocol to differentiate between PEDV and TGEV directly from formalin-fixed, paraffin-embedded tissue, using a multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. (omitted)

• 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.

• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2002.11a
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• pp.143-143
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• 2002

돼지 장염 바이러스를 동시에 감별 진단할 수 있는 새로운 진단법인 oligonicleotide microarray 기법을 돼지 설사 분변을 대상으로 바이러스 감염을 진단하고 기존의 진단법으로 널리 사용되고 있는 RT-PCR과 진단기법의 민감도와 특이도를 비교하고자 하였다. 32개 양돈장, 102 예의 포유 및 이유자돈의 설사분변에서 microarray를 이용한 검사결과 Transmissible gastroenteritis virus (TGEV) 9.8％, porcine epidemic diarrhea virus (PEDV) 28％, (porcine enteric calicivirus (PECV) 18.6％, porcine rotavirus (PRV) group A 6.9％, PRY group C 1％ 의 검출률을 확인하였다. 또한 RT-PCR 기법과의 비교에서도 100％의 민감도와 72.2％의 특이도를 보였으며 agreement는 85.3％, kappa value 0.71로 우수한 진단기법임을 확인하였다. 이를 통해 microarray 진단법은 RT-PCR 후의 전기영동 과정과 민감도를 높이기 위해 수행되는 nested PCR 수행의 번거러움을 없애면서 정확한 감별진단을 수행할 수 있는 진단 기법임을 확인하였다.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.5
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• pp.527-533
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• 2017

We investigated the infection of nervous necrosis virus (NNV) in seven band grouper Hyporthodus septemfasciatus broodstocks, which have been reared in aquaculture farms in South Korea during 2012-2014. To investigate the prevalence of NNV within the broodstock, egg, sperm, and blood were sampled in the spawning season. The egg and sperm samples were subjected to a nested reverse transcription (RT) polymerase chain reaction (PCR) assay to detect NNV and were inoculated on SSN-1 cells to culture the virus. Blood samples were used to detect antibodies against NNV using enzyme linked immunosorbent assay (ELSIA). Positive values from ELISA were found in 39 of 162 samples (24%) in 2012, and 13 of 28 samples (46%) in 2014. Additionally, 4 of 34 broodstocks (11%) investigated in 2013-2014 were determined to be carriers from the nested RT-PCR and in vitro cultivation. The broodstocks in which antibodies against NNV were detected by ELISA, or in which NNV was detected by the nested RT-PCR assay, posed a risk of vertical transmission of NNV. Therefore, it is necessary to select virus-free broodstocks in seed production to reduce the possibility of the vertical transmission of NNV.