• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• Korean Journal of Poultry Science
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• v.44 no.2
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• pp.93-102
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• 2017

A duplex RT-PCR (dRT-PCR) assay was developed for the simultaneous detection and discrimination of non-virulent and virulent Newcastle disease virus (NDV) in a single PCR tube. Primers targeting the large polymerase protein (L) gene and the fusion protein (F) gene of NDV were designed to detect all NDVs (by common type PCR primers) and virulent NDVs (by pathotype PCR primers), respectively and evaluated experimentally with reference NDV strains and other poultry viral pathogens. PCR products of the expected size of 386 bp were amplified from all NDV samples whereas PCR products of the expected size of 229 bp were amplified from virulent NDV samples alone. Cross reaction was not observed with other avian viral pathogens. The detection limit of NDV by the dRT-PCR was estimated to be $10^3$ 50% egg infectious dose/0.1 mL. In the dRT-PCR using field isolates of NDV, the pathotype PCR primers detected specifically all of virulent field isolates of NDV from Malaysia, Pakistan and China whereas common type PCR primers detected 94.4% (51/54) of field isolates of NDV from China. Three Chinese NDV isolates with false negative result were non-virulent viruses. Our results indicate that the dRT-PCR might provide a rapid and simple tool for rapid simultaneous detection and discrimination of non-virulent and virulent NDVs. Therefore the developed dRT-PCR assay provides a powerful novel means for the rapid diagnosis of Newcastle disease.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.198-204
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• 2002

Poliovirus is a member of enterovirus which causes paralytic poliomyelitis, encephalitis and aseptic meningitis. Since poliovirus is spread by the fecal-oral route and poliovirus-contaminated water could be a potential threat for public health, detection of poliovirus in drinking water resource is important. Infectious poliovirus and poliovirus inactivated by heat or UV were used to test three detection methods such as cell culture method, reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture (ICC)-PCR. Infectious poliovirus was detected by all three methods and ICC-PCR was the most sensitive and fast in detecting poliovirus. Inactivated polioviruses could not be detected by cell culture or ICC-PCR methods. On the other hand, heat- inactivated viruses could be detected by RT-PCR. Thus it is suggested that ICC-PCR method is the most sensitive and effective in detecting infectious polioviruses in water sample.

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Journal of Microbiology
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• v.38 no.4
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• pp.260-264
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• 2000

Reverse transcription (RT)-PCR and nested PCR methods (2-step PCR) were tested for their ability to detect marine birnavirus (MBV) in cultured rockfish, Sebastes schlegeli. One set of primers for RT-PCR was designed, based on a gene of infectious pancreatic necrosis virus (IPNV), and another set of primers for nested PCR was designed based on the VP2/NS junction region of MBV. This 2-step PCR method was specific for MBV and sensitivity was heightened when nested PCR was combined to RT-PCR. This 2-step PCR method was useful for detecting MBV not only in diseased fish, but also in asymptomatic fish. These results indicate that this 2-step PCR method is useful for detecting MBV in rockfish.

• Biomedical Science Letters
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• v.21 no.4
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• pp.227-232
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• 2015

Epstein-Barr virus (EBV) has a pathogenic role in several lymphomas including diffuse large B-cell lymphoma (DLBCL). In this study, we detected EBV from formalin-fixed paraffin embedded (FFPE) tissues of DLBCL patients by RT-PCR and compared the sensitivity of the RT-PCR method to in situ hybridization (ISH) method. The RNA was extracted from 91 FFPE samples with DLBCL and amplified with primers targeting EBV-encoded small RNA (EBER) by RT-PCR. When using the RT-PCR method, 13 of 91 patients (14.3%) were positive and among these 13 cases, 7 cases (7.7%) were from > 50-year-old patients that is classified as EBV positive DLBCL of the elderly. In previous results using ISH method, 3 of 91 patients (3.3%) were positive and 2 case (2.4%) were older than 50-year-old. These results indicate that RT-PCR method used in this study shows a higher sensitivity than ISH method. The ratio of male versus female among the EBV positive samples was 1.2:1 with the ratio of male higher. If RT-PCR method having high sensitivity is used simultaneously as well as the ISH method providing the information of the EBV positive cellular location, it is expected that EBV will be more accurately detected.

• Research in Plant Disease
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• v.13 no.2
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• pp.82-87
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• 2007

The genetic diagnostic method of virion capture (VC)/RT-PCR was developed for the simultaneous detection of three rod shaped viruses of Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus(KGMMV) and Zucchini green mottle mosaic virus (ZGMMV) transmitted by seed in Cucurbit. Out of 12 primer combinations for the three tobamoviruses, a primer set of CGMMV-C724, KGMMV-K513 and ZGMMV-Z407A was useful for mono and triplex VC/RT-PCR. The triplex VC/RT-PCR for the three tobamovirus in Cucurbit could detect specifically without interference among primers and/or plant species of watermelon, gourd, cucumber, melon, pumpkin, squash and Nicotiana benthamiana.

• The Plant Pathology Journal
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• v.27 no.1
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• pp.44-52
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• 2011

Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.339-347
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• 2012

Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.