• Journal of Apiculture
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• v.32 no.2
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• pp.119-131
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• 2017

For the Rapid detection of small hive beetle (SHB; Aethina tumida) and for the mass-survey against SHB invasion, SHB-specific ultra-rapid PCR system was developed. Three different pairs of Aethina tumida-specific primers were deduced from cytochrome oxidase subunit I (COI) gene in mitochondrial DNA of SHB. Using optimized SHB-specific ultra-rapid PCR, $2.1{\times}10^1$ molecules of COI gene belonged to SHB could be detected specifically and quantitatively within 18 minutes 40 seconds. For the purpose of the application in apiary field, a DNA extraction method from bee debris was separatedly developed. When $10^5$ SHB-specific COI molecules (1/1000 body of SHB larvae) are existed in 1g of bee debris, it could be verified inner 10 minutes as qualitative and quantitative manner. SHB-specific ultra-rapid PCR we proposed would be expected to apply widely, either in apiary field or laboratory, for the rapid detections and the control against SHB-invasion.

• 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.

• Journal of Apiculture
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• v.34 no.1
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• pp.39-46
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• 2019

BQCV multi-point PCR was developed as a rapid multiplex detection method for BQCV, one of the viral pathogens of honeybees. It could detect BQCV specific genes qualitative as well as quantitative detection based on ultra-rapid PCR. Three primer pairs (RNA dependent RNA polymerase, capsid protein, 3C like protease) were specifically designed for accurate the detection and were optimized for minimizing the detection time and increasing the sensitivity. Our advanced diagnostic system have the accuracy by lowering the concern about the variation in the BQCV detection site. In addition, it should be an opportunity to identify mutations that are mixed with other viruses.

• Journal of Apiculture
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• v.34 no.1
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• pp.15-26
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• 2019

Tracheal mite (Acarapis woodi) is an internal parasite that is parasitic on the bronchus of adult bees and sucks fluid from the trachea. Since its first report by Rennie, it has been spread throughout Europe and in some Asian regions, with adjacent Japan and China reported in 2011 and 2012, respectively. Korea detected specific genes of A. woodi in 2015, but only one of 99 samples has been identified and the being of A. woodi has not been confirmed. In this study, we established a specific nested PCR method to confirm for detecting low-copy number of A. woodi-specific gene in bee samples. As a result, A. woodi-specific COI gene was amplified in 15 of 23 samples, and they were judged positive by melting point analysis and sequencing analysis. Although we could not observe the existence of the mites in bees, our results suggest that tracheal mit might exist in nature.

• Journal of Apiculture
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• v.34 no.1
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• pp.27-37
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• 2019

Rapid detection of Tropilaelaps, an external parasite of honeybees that lead to malformation of honeybee or colony collapse disorder, is becoming important. But it is very difficult to find with the naked eye of Tropilaelaps. In this study, we have developed a method to detect the specific gene of Tropilaelaps from the hive debris and to know the number of Tropilaelaps in the hive through Tropilaelaps-specific quantitative detection. Tropilaelaps-specific gene amplified in DNA extracted from hive debris by consecutive PCR (1st detection, 2nd nested PCR). It could detect 101 molecules level of Tropilaelaps-specific gene and confirm the amplification of the Tropilaelaps-specific gene. It was possible to accurately quantify the number of Tropilaelaps from the hive debris sample, which is difficult to discriminate the presence of Tropilaelaps visually, through Tropilaelaps-specific detection. Under the microscope, Tropilaelaps was collected and quantitative detection of Tropilaelaps-specific genes was performed. It was possible to quantify the number of Tropilaelaps present in the hive through the molecules of the quantified Tropilaelaps-specific genes. We suggest that hive debris can represent as a micro-environment to hive and show that it can be a simpler and more accurate sample than using a parasitic host honeybee. We expect that hive debris should facilitate the monitoring of Tropilaelaps in hive.