Expression of RNA dependent RNA polymerase gene from BQCV in honey bee for application of monoclonal antibody generation

Giang Huong Thi Luong,Byoungsu Yoon 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Black queen cell virus (BQCV), one of the most prevalent viruse, causes the death of queen larvae and pupae. The RNA-dependent RNA polymerases (RdRPs) are central components in the life cycle of RNA viruses that catalyzes the replication of RNA from an RNA template without DNA stage. Inhibition of RdRP gene is importantly significant for application of monoclonal antibody generation as a diagnosis tool for identifying BQCV infection in honey bee..In this study, the presence of BQCV in honey bee samples was confirmed by PCR using BQCV F/R primer set to multiply of 700 bp DNA fragment. For ampification of BQCV Rdrp gene, a primer set attached BamHI/SalI restriction site was designed based on the best homogenization between BQCV RdRP sequences in NCBI, a PCR product containing BQCV RdRP gene with 1576 bp in length was amplified. Furthermore, BQCV RdRP gene will be cloned into pBlueXcm vector for future researches.

Loop-mediated Isothermal Amplification(LAMP) 법을 이용한 Kakugo Virus의 검출법 개발

이중구,윤병수 경기대학교 사회과학연구소 2012 경기대학교 기초과학논문집 Vol.25 No.1

Kakugo Virus(KV) is a picorna-like virus that was originally identified in the brains of aggressive but apparently healthy worker honeybees(Apis mellifera L.). A loop-mediated isothermal amplification (LAMP) assay allows one-step detection of gene amplification without expensive equipment such as thermocycler. In this study, new LAMP method for the specific detection of KV in honeybee was developed (KV-LAMP). A set of four designed primers named KV-F3 / B3 / FIP / BIP that recognize KV sepcific sequence(GenBank, 250362.1). After optimization of conditions, KV-LAMP specific amplification was successfully performed using KV-LAMP from standard DNA sequence. KV-specific template was amplified under isothermal conditions at 59℃ within 60 minutes by KV-LAMP. The method could be detected KV from DNA clone at 1×102copies/㎕, also could be applied not only cDNA but also field samples. Especially, this method was developed direct detection by the naked eye using SYBR Green Ⅰ and Gene-Finder™ Nucleic acid fluorescent dye. In conclusion, KV-LAMP may be a useful method for the detection Kakugo Virus from the field. Kakugo Virus(KV)는 외관상으로는 건강하지만 공격적인 성향을 나타내는 일벌의 뇌에서 분리된 picorna virus 계열의 것이다. LAMP는 thermocycler 같은 고가의 장비없이도 유전 자를 증폭할 수 있는 등온증폭방법으로, 본 연구에서는 쉽고 빠르게 KV를 진단할 수 있는 KV-LAMP법을 개발하였다. KV의 특이적인 염기서열(GenBank, AB250362.1)에 근거한 KV-F3 / B3 / FIP / BIP, 4개의 primer를 사용하여 59℃의 등온조건하에서 60분간 정치하여 DNA가 성공적으로 증폭됨을 확인하였다. 1×102copies/㎕까지 증폭이 가능하며 실제 꿀벌에서도 검출가능함이 확인되었고, SYBR GreenⅠ과 Gene-Finder™ Nucleic acid fluorescent dye를 사용하여 전기영동 없이 육안으로 관찰할 수 있었다. 따라서 KV-LAMP 법을 통하여 KV를 신속하고 정확하게 검출함으로써 현장에서 유용하게 적용될 수 있을 것 으로 사료된다.

동일 상황의 검도경기에서 31인의 심판들 간 판정견해의 차이

박학진(Hak-Jin Park),윤병수(ByoungSu Yoon) 대한검도학회 2005 대한검도학회지 Vol.21 No.1

검도경기 중 여러 개별적 상황에 대하여 31명의 국내 최고 수준의 심판들의 판정 간 차이점과 다양성을 조사하였다. 이 분석을 위하여 추가적으로 31명의 심판들은 각 상황에 대하여 자신이 판정에 이르기까지의 과정을 정확하게 설명하였다. 결과로써, 검도경기 중 여러 선별된 상황들에 대하여, 같은 상황임에도 유의하게 다른 판정의견들이 있음이 발견되었다. 특히, 정면머리의 유효격자에 대하여 크게 다른 물리적 척도가 심판 간에 있음을 나타나게 되었고, 동일한 금지행위 및 벌칙 조항에 근거한 용인될 수 없는 상이한 판정이 있음이 발견되었다. 각기 다른 판정의 합리적 합의를 위하여 반대의견을 가진 각 심판진의 의견들이 논의되었다. On the same situations in Kumdo match, the variations of judgements among the 31 korean referees were investigated, who are all belong to one of best selection of korean referees. For this analysis referees exactly explained additionally a process of making his judgement on 19 individual situations in Kumdo match. As the results. on some selected situations in Kumdo match, significantly different opinions of judgements in same situation were found. Especially, it was revealed that there are much different physical measures on the valid strike (Yukv-datotsu) of the forehead (Men-bu) among the individual judgement. It was also found that there were unacceptably different judgements based on identical acts of prohibition and penalties. On each variation of judgement, both opinions of referee s group were discussed for the rational agreement.

Development of PCR method for distinguishment of Sacbrood virus (SBV) based on SBV nucleotide acid sequence

Joong-Goo Lee,Hee-Young Lim,Byoungsu Yoon 한국양봉학회 2014 한국양봉학회 학술대회 자료집 Vol.2014 No.4

검도경기 중 같은 상황에 대한 심판 간 판정의 차이에 관한 연구

박학진(Hak-Jin Park),윤병수(ByoungSu Yoon) 대한검도학회 2004 대한검도학회지 Vol.20 No.1

국문요약 검도경기 중 여러 개별적 상황에 대하여 10명 국내 심판들의 판정 간 차이점과 다양성을 조사하였다. 이 분석을 위하여 국내 최고 수준의 심판들이 선별되었으며, 10명의 심판들은 각 상황에 대하여 자신이 판정에 이르기까지의 과정을 정확하게 설명하였다. 결과로써, 검도경기 중 여러 선별된 상황들에 대하여, 같은 상황임에도 유의하게 다른 판정의견들이 있음이 발견되었다. 특히, 정면머리의 유효격자에 대하여 크게 다른 물리적 척도가 심판 간에 있음을 알게 되었고, 동일한 금지행위 및 벌칙 조항에 근거한 용인될 수 없는 상이한 판정이 있음을 발견하였다. 각기 다른 판정에 대하여 합리적 합의를 위하여 심판진의 양쪽 의견이 논의되었다. The differences and variations of judgements by the 10 korean referees on the some individual situations in Kumdo match were investigated. For this analysis ten referees exactly explained a process of making his judgement on each situation in Kumdo match, who are all belong to one of best group of korean referees. As the results, on some selected situations in Kumdo match, significantly different opinions of judgements in same situation were found. Especially, it was known that there are much different physical measures on the valid strikes(Yuko-datotsu) of the forehead(Men-bu) among referees. It was also found that there were unacceptably different judgements based on identical acts of prohibition and penalties. On each different judgement, both opinions of referee's group were discussed for the rational agreement.

Molecular Detection of Honey Bee Pathogenic Microbes: Recent Advances and Future Perspective

Nguyen Thi Kim Cuc,Nguyen Van Phu,ByoungSu Yoon 한국양봉학회 2021 韓國養蜂學會誌 Vol.36 No.2

Microbes, including bacteria and viruses, are the main threats to the health of honey bee colonies, and cause great losses to beekeepers. Rapid and accurate diagnosis is the key to controlling and eliminating honey bee diseases, and preventing them from spreading and causing an outbreak. This review summarizes recent advances in techniques to detect honey bee diseases, including traditional methods such as polymerase chain reaction, and next-generation sequencing methods, and how they are applied in the diagnosis and management of such diseases. It also discusses how these methods have revolutionized disease detection, and presents the future directions in the field of clinical diagnostics.

꿀벌세포의 세포배양에 적합한 성장 배양액 선별

주현희(Hyunhee Ju),길성호(Sungho Ghil),Byoungsu Yoon 한국양봉학회 2013 韓國養蜂學會誌 Vol.28 No.5

Here, we select compatible cell growth media for cell culture of honey bee, Apis mellifera. Eggs of honey bee harvested from bee comb were sterilized by using sterilization buffer. Sterilized eggs were dissected with three different growth media; (1) growth media A containing Graces insect media, (2) growth media B containing Hinks TNM-FH media, and (3) growth media C containing Schneiders insect media. Each media were used as basal media for cell culture. After dissection of eggs with pipetting, the cells were seeded into tissue culture dishes. When we used growth media A and B, the cells did not adhere to bottom of tissue culture dish and all cells were not shown at 8 days due to cell death. However, interestingly, when we used growth media C, the cells adhered to bottom of tissue culture dish and grew with fibroblast-like morphology until 8 days, suggesting that the compatible growth media for cell culture of honey bee is Schneiders insect media in our culture system. We consider that further experiments are needed to increase cell viability and proliferation.

불량꿀 검사를 위한 개선된 분석 방법에 관한 연구

유응철(Eung-Cheol Yoo),공영건(Young-Kun Kong),윤병수(ByoungSu Yoon) 한국양봉학회 2010 韓國養蜂學會誌 Vol.25 No.1

Natural honey shall not have added to it any ingredient, nor shall be made to be spiked with sugars other than nectar, due to its natural origin and genuine purity. Already it was well known that many adulterated honey could be passed in the authentic test using EA-IRMS (Elemental Analysis - Isotope Ratio Mass Spectrometry) method. Adulteration of honey may be increased, because simple C13/C12 isotope ratio of total honey could be easy adjusted by adding or spiking of sugars. Therefore, improved analysis-method are urgently required for the honey authenticity to detect and prevent adulteration of honey. LC-IRMS (Liquid Chromatography-Isotope Ratio Mass Spectroscopy), or LC coupled to high-precision IRMS, has been available commercially since 2004. To improve isotopic methods devoted to honey authenticity, new procedure using LC-IRMS was developed on 2006. In this study, advantages and disadvantages of EA-IRMS and LC-IRMS were discussed. From what adulteration were happened in Korea, we concluded that LC-IRMS method should be adopted to the authentic test of honey, after sufficient test for technical qualifications.

Loop-mediated Isothermal Amplification(LAMP) 법을 이용한 Sacbrood Virus (SBV)의 검출법 개발

이보람(Boram Lee),노지나(Ji-Na No),유미선(Mi-Sun Yoo),윤병수(Byoungsu Yoon) 한국양봉학회 2011 韓國養蜂學會誌 Vol.26 No.4

Sacbrood virus (SBV) is an infectious disease which affects the brood of honeybees, resulting in failure to pupate and death. A loop-mediated isothermal amplification (LAMP) assay allows one-step detection of gene amplification without expensive equipment such as thermocycler. The SBV-LAMP method was developed for detection of SBV easily and rapidly. A set of four designed primers named SBV-F3/B3/FIP/BIP that recognize SBV specific sequence. After optimization of conditions, SBVspecific amplification was successfully performed using SBV-LAMP from standard. SBV-specific template under isothermal conditions at 57°C within 60 minutes. The method could be detected SBV in DNA clone at 103copies/μl, also could be applied not only cDNA but also field samples. Especially, this method was developed direct detection analysis by the naked eye using SYBR Green I, Gene-FinderTM Nucleic acid fluorescent dye and phenol red. SBV-LAMP may be expected to be useful for the specific detection of SBV in field and for the monito ring of natural infection in Apis mellifera L. by SBV.

Identification of Kakugo Virus in Honeybees from Korea

Boram Lee,Phu Van Nguyen,Soon-Bok Lee,Byoungsu Yoon 한국양봉학회 2012 韓國養蜂學會誌 Vol.27 No.1

Kakugo virus (KV) is a picorna-like virus that was originally identified in the brains of aggressive but apparently healthy worker honeybees (Apis mellifera L.). This is the first report of KV infection in Korea. Infection with KV was identified in Korean honeybees by PCR. The sequence of the PCR product was determined and analyzed by T-vector cloning. Homology between the determined and reported KV sequences were calculated, and the results showed a 96.6% similarity for the nucleotide sequence and 99.3% amino acid sequence homology. The PCR method was able to easily detect KV in honeybees. Here, we report the specific KV primer pairs for the detection and verification of KV infection.