• Journal of Apiculture
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• v.32 no.2
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• pp.99-109
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• 2017

The multiple real-time PCRs against pathogens of Bombus species including DWV, IAPV, KBV, SBV, BQCV, kSBV, SBPV and Paenibacillus larvae, Mellisococcus plutonius, Lysinibacillus fusiformis, and Klebsiella oxytoca have been developed. One extracted nucleic acid from Beesample could be applied to 11 different PCRs in same time and condition. Specific PCR-products were amplified qualitative and quantitative manner inner 20 minutes successfully, when each 1000 molecules of pathogen-specific target DNA is existed as template, respectively. The multiple PCR detection that we propose would be expected to apply to quarantine test for international exchange of Bombus species.

• Journal of Life Science
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• v.26 no.5
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• pp.620-631
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• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.

• Food Science of Animal Resources
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• v.33 no.5
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• pp.610-616
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• 2013

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.

• Biomedical Science Letters
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• v.6 no.1
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• pp.73-82
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• 2000

Blunt-end DNA fragments can be inserted in two different orientations. Conventionally, their directions are determined by restriction enzyme digestion or by DNA sequencing, however, these methods are often limited in their use due to the lack of appropriate enzyme sites or large sample numbers, respectively. In the present study, a novel strategy and the corresponding protocol for the simple determination of insert orientation is introduced. Using conventional sequencing primers and PCR primers that have been used for amplification of the insert, single clones, which have inserted the fragment in the desired orientation, were easily identified by this PCR-based method. The fidelity of this system was confirmed by cloning of a tar urocortin cDNA, which is a recently discovered neuropeptide. Recombinant clones identified by this method were further shown to be fully functional, and using these, for the first time, urocortin was recombinantly expressed in eukaryotic cells.

• 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.

• Journal of fish pathology
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• v.12 no.1
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• pp.49-55
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• 1999

To detect birnavirus from cultured marine fish, RT-PCR assay was developed. This method was specific for aquatic birnaviruses that include IPNV Sp., IPNV Ab, IPNV VR-299 and MBV Y6. The birnavirus gene was detected (birnavirus positive samples detected 46/50) from clinical samples signed with abdominal distension and overall darkening even though the samples gave negative results in virus isolation (birnavirus isolate with CHSE-214 cell showed 12/50).

• Biomedical Science Letters
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• v.5 no.1
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• pp.131-133
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• 1999

In order to the investigate epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA), 31 strains of Staphylococcus aureus were isolated from the equipments of two hospitals in Chonbuk. And their antimicrobial resistance patterns against 7 kinds of antimicrobial agents and the identification of MRSA by polymerase chain reaction (PCR) were studied. Seven strains among 10 strains of methicillin resistant Staphylococcus aureus showed 554 bp DNA which was a part of mecA gene in PCR analysis.

• Journal of the Korean Chemical Society
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• v.52 no.6
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• pp.630-636
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• 2008

• Korean Journal Plant Pathology
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• v.12 no.4
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• pp.432-436
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• 1996

역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 담배(Nicotiana glutinosa)에 증식시킨 7종의 오이 모자이크 바이러스(CMV)를 검정하였다. RT-PCR을 위한 간단하고 신속한 바이러스 핵산의 조즙액 추출법이 개발되었으며, CMV 외피단백질 유전자 부위를 기초로 하여 제작한 20개의 염기로 구성된 primer를 사용하여 RT-PCR을 실시한 결과, 약 490 염기쌍의 DNA 단편들이 이병식물의 조즙액으로부터 증폭되었다. EcoRI 및 MspI을 이용한 RT-PCR 산물의 분석에 의하여, 공시한 7종의 바이러스는 모두 CMV subgroup I으로 동정되었다. Ouchterlony 한천젤 이중 확산법을 이용한 항혈청 검정에서도 7종의 바이러스 모두 CMV-Y의 항혈청과 단일의 침강선을 형성하였다.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.19-27
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• 1997

To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within env gene of SRV subtype 1 (SRV-1). The 3' proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targeting an env region successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-Iymphotropic virus type 1, major immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than $5-7{\times}10^4$ uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening system with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.