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기문응애(Acarapis woodi) 특이 유전자 검출을 위한 초고속 nested PCR법 개발
김문정(MoonJung Kim),김병희(Byoung-Hee Kim),김소민(SoMin Kim),Truong A Tai,김정민(Jung-Min Kim),김선미(Seonmi Kim),윤병수(Byoung-Su Yoon) 한국양봉학회 2019 韓國養蜂學會誌 Vol.34 No.1
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.
Acute Bee Paralysis Virus (ABPV)의 정확한 검출을 위한 Nested 초고속 PCR법 개발
김문정(MoonJung Kim),김정민(JungMin Kim),김병희(ByeongHee Kim),김소민(SoMin Kim),Truong A Tai(A Tai Truong),윤병수(ByoungSu Yoon) 한국양봉학회 2018 韓國養蜂學會誌 Vol.33 No.3
Acute bee paralysis virus (ABPV) is an infectious disease causing paralysis in healthy honeybee colonies. Because ABPV is highly homologous with relative viruses, it is difficult to distinguish ABPV from especially IAPV or KBV by molecular diagnosis. In this study, for accurate and specific detection of ABPV, a secondary nested PCR detection method following the primary detection PCR against ABPV was developed. It could be able to detect from less than 100 molecules of ABPV. The detection time was also minimized from RNA extraction to PCR. Using ultra-rapid ABPV-specific detection and nested PCR, the presence of ABPV was detected from infected bee samples within 50 minutes. It might be expected that this kind of detection would be helpful for accurate detection and monitoring of ABPV in environment.
사탕수수 설탕 및 사양꿀에서 사탕수수 (Saccharum officinarum) 고유 유전자의 검출
김병희(Byounghee Kim),김소민(Somin Kim),김문정(Moonjung Kim),김정민(Jungmin Kim),Truong A Tai(A Tai Truong),윤병수(Byoungsu Yoon) 한국양봉학회 2018 韓國養蜂學會誌 Vol.33 No.3
Sugar cane-specific gene could be successfully amplified with DNAs isolated from sugar or sugarhoney 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.
사탕무(Beta vulgaris) 설탕으로 제조된 사양꿀에서 사탕무 고유 유전자의 검출
김소민(Somin Kim),김병희(Byounghee Kim),김문정(Moonjung Kim),김정민(Jungmin Kim),Truong A Tai(A Tai Truong),윤병수(Byoungsu Yoon) 한국양봉학회 2018 韓國養蜂學會誌 Vol.33 No.3
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 URqPCR, sugar beet-specific DNA sequence could be amplified quantitatively until 10<SUP>0</SUP> 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.
Multi-point PCR법을 이용한 Black Queen Cell Virus (BQCV) 검출법 개발
김소민(Somin Kim),김병희(Byounghee Kim),김문정(Moonjung Kim),김정민(Jungmin Kim),Truong A Tai,김선미(Seonmi Kim),윤병수(Byoungsu Yoon) 한국양봉학회 2019 韓國養蜂學會誌 Vol.34 No.1
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.
Rapid detection of viral RNA in honeybee using ultra-rapid qPCR and a DNA chip
Jung-Min Kim,Su-Jin Lim,SoMin Kim,MoonJung Kim,ByoungHee Kim,Truong A Tai,김선미,ByoungSu Yoon 대한수의학회 2020 Journal of Veterinary Science Vol.21 No.1
The emergence and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in livestock animals have become a worldwide public health concerns. While the prevalence and genetic profiles of MRSA strains in pigs and pork meat have been actively studied, livestock-associated MSSA strains have only been characterized in a few small-scale studies. In this investigation, we assessed the nationwide prevalence of MSSA in the Korean pig production chain, including pig farms, slaughterhouses, and retail markets. Among the 41 MSSA strains, the predominant clonal lineages were sequence type (ST) 398 (n = 15, 37%) and ST5 (n = 13, 32%). Although the overall prevalence of MSSA (2.58%) was low and mostly restricted to pig farms, ST398 MSSA strains showed higher level of multidrug resistance phenotype versus non-ST398 MSSA strains. In addition to the MDR phenotype, all of the ST398 MSSA strains exhibited resistance to tetracycline as they harbored the tet(K), tet(L), and/or tet(M) genes. However, ST398 MSSA strains did not exhibit increased resistance to zinc compared with the non-ST398 strains. This study is the first to provide evidence of ST398 MSSA emergence in livestock animals in Korea. Further studies are necessary to elucidate the potential of ST398 MSSA strains for human transmission. Our findings suggest that the MDR phenotype and high levels of tetracycline resistance may have played an important role in the emergence and prevalence of ST398 MSSA in pig farms in Korea.
봉변에서 특이 유전자 검출법에 의한 봉군 내 꿀벌가시응애류 (Tropilaelaps)의 정량적 검출
김병희(Byounghee Kim),김소민(Somin Kim),김문정(Moonjung Kim),김정민(Jungmin Kim),Truong A Tai,김선미(Seonmi Kim),윤병수(Byoungsu Yoon) 한국양봉학회 2019 韓國養蜂學會誌 Vol.34 No.1
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 10<SUP>1</SUP> molecules level of Tropilaelapsspecific 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.
Truong, A.-Tai,Kim, Byounghee,Kim, Somin,Kim, Moonjung,Kim, Jungmin,Kim, Seonmi,Yoon, Byoungsu International Bee Research Association [etc.] 2019 Journal of apicultural research Vol.58 No.5
<P> Israeli acute paralysis virus (IAPV) is one of the main pathogens involved in the collapse of honey bee colonies. However, because the virus exhibits a high level of genetic variation and some IAPV strains exhibit high degrees of homology with related viruses, the detection of IAPV in infected honey bees is relatively challenging. To address this obstacle, the aim of the present study was to develop a new detection method that relies on multiple detection sites within the IAPV genome and on Ultra-rapid real-time polymerase chain reaction (UR-qPCR). The new system simultaneously targeted a RNA-dependent RNA polymerase gene <I>(RdRp)</I> and two capsid genes (<I>VP3</I> and <I>VP1</I>). This multi-point PCR approach was highly efficient, with the ability to detect 100% of IAPV infections, and outperformed single-point PCR, which was only able to detect 86.96-95.6% of IAPV infections. Sequence analysis indicated that <I>RdRp</I> was more variable than the two capsid genes, and the specificity of the proposed method was demonstrated by the detection of IAPV from samples co-infected by IAPV and KBV. Importantly, both freezing-thawing RNA isolation and UR-qPCR could be performed in 27min and 40s. Therefore, the present provides an useful tool for the rapid identification of IAPV in apiaries. </P>