• Title, Summary, Keyword: Ultra-rapid PCR

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Multi-Ultra-Rapid PCR against Viral Pathogens of Honeybee using Hive Debris (봉변을 이용한 꿀벌 바이러스성 병원체의 다중 초고속 PCR법)

  • Kim, Jung-Min;Truong, A Tai;Kim, So-Min;Kim, Byoung-Hee;Kim, Mun-Jung;Yoon, Byoung-Su
    • Journal of Apiculture
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    • v.33 no.3
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    • pp.135-147
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    • 2018
  • Hive debris is a kind of micro-environment of beehive including excreta, mucus, epidermis of bee and carcass. Hive debris might also include almost invaded pathogens and particle of hive parasite and injurious insect against honeybee Multi-Ultra-Rapid PCR (8MUR-PCR) against eight viral pathogens in honeybee, including black queen cell virus, deformed wing virus, sacbrood virus, korean sacbrood virus, acute bee paralysis virus, chronic bee paralysis virus, slow bee paralysis virus and israeli acute paralysis virus, were optimized for one PCR condition. Using several hive debris from Suwon province, 8MUR-PCR was applied and detection-results were analyzed. In many cases, viral pathogens were found more accurately from hive debris than from bee carcass itself, even both originated from same bee-population. Based on this results, hive debris might be better than pathogenic samples for the general survey of public hygiene in single population.

Development of mcyB-specific Ultra-Rapid Real-time PCR for Quantitative Detection of Microcystis aeruginosa (Microcystis aeruginosa의 정량을 위한 mcyB 특이 초고속 실시간 유전자 증폭법의 개발)

  • Jung, Hyunchul;Yim, Byoungcheol;Lim, Sujin;Kim, Byounghee;Yoon, Byoungsu;Lee, Okmin
    • Journal of Korean Society on Water Environment
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    • v.34 no.1
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    • pp.46-56
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    • 2018
  • A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

Detection of Sugar Cane (Saccharum officinarum)-specific Gene from Sugar and Sugar-honey (사탕수수 설탕 및 사양꿀에서 사탕수수(Saccharum officinarum) 고유 유전자의 검출)

  • Kim, Byounghee;Kim, Somin;Kim, Moonjung;Kim, Jungmin;Truong, A Tai;Yoon, Byoungsu
    • Journal of Apiculture
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    • v.33 no.3
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    • pp.221-226
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    • 2018
  • Sugar cane-specific gene could be successfully amplified with DNAs isolated from sugar or sugar-honey using Saccharum officinarum-specific Ultra-Rapid or conventional PCR. Specificity of PCR products from sugar or sugar-honey was verified by nested PCR and DNA sequencing. This PCR could be applied to a quantitative analysis for honey-evaluation. In our knowledge, it is first report that sugar cane-specific sequence could be detected from sugar-honey or sugar itself, and that sugar-honey could be evaluated by genetic examination.

Detection of Sugar Beet (Beta vulgaris) - Specific Gene from Honey Made by Sugar of Sugar Beet (사탕무(Beta vulgaris) 설탕으로 제조된 사양꿀에서 사탕무 고유 유전자의 검출)

  • Kim, Somin;Kim, Byounghee;Kim, Moonjung;Kim, Jungmin;Truong, A Tai;Yoon, Byoungsu
    • Journal of Apiculture
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    • v.33 no.3
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    • pp.213-219
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    • 2018
  • We could detect the specific gene of sugar beet (Beta vulgaris) from honey produced by sugar of sugar beet using the ultra-rapid real-time PCR (UR-qPCR). In our knowledge, it is the first report in the world that PCR detection of sugar beet-specific gene from adulterated honey. Through extracting the DNA (using CTAB method) from sugar and honey of sugar beet and using the UR-qPCR, sugar beet-specific DNA sequence could be amplified quantitatively until $10^0$ molecules of initial template. By using nested PCR and DNA sequencing, its specificity was confirmed. DNA sequence was matched 100% with mitochondria gene of sugar beet. This finding that trace DNA in adulterated honey could be genetically analysed, would be used as a decisive evidence for the authenticity test of honey.