Development of methods for accurate detection of honeybee pathogens and molecular determination of adulterated honey

Thesis for Degree of Doctor Development of methods for accurate detection of honeybee pathogens and molecular determination of adulterated honey by Truong A Tai Department of Life Science Graduate School Kyonggi University (2019) Development of methods for accurate detection of honeybee pathogens and molecular determination of adulterated honey A dissertation submitted to the faculty of Graduate School of Kyonggi University in the fulfillment of the requirement for the Degree of Doctor of Philosophy December 2019 Graduate School of Kyonggi University Department of Life Science Truong A Tai Contents List of Tables ······································································································x List of Figures ··································································································xii Abstract ··············································································································xvi Part Molecular characterization of honeybee pathogens ··········1 Chapter Genotyping quantification of Sacbrood virus ··············1 I Introduction ····························································································1 Sacbrood virus (SBV) ····································································1 Genotypes of Sacbrood virus ·······················································2 Detection of SBV ············································································3 Purpose of this study ····································································3 II Materials and methods ······································································4 Bacterial strain and plasmid vector for molecular cloning Bacterial culture medium ······························································5 SBV-infected honeybee samples ·················································6 Plasmid DNA isolation ···································································7 Total RNA isolation and standard DNA construction ··········7 Primer design ···················································································8 - i - Reverse transcription ····································································11 Standard DNA construction ·······················································12 Specific identification of genotyping DNA ·····························13 10 Sensitivity of SBV detection ····················································13 11 Quantitative detection of SBV genotypes ····························14 12 Agarose gel electrophoresis ······················································15 III Results and discussion ···································································15 Standard DNAs for SBV genotyping ······································15 Sensitivity of genotyping in single PCR and nested PCR 17 Accuracy of SBV genotyping on standard DNAs ···············19 Detection of SBV genotypes from honeybee samples ········21 Quantification of SBV genotypes ·············································23 IV Conclusion ·························································································24 Chapter Evaluation of point mutation on the minor capsid protein (MiCP) gene of Sacbrood virus ··········································26 I Introduction ··························································································26 II Materials and methods ····································································27 Source of SBV nucleic acids ·····················································27 Detection of SBV ··········································································27 Molecular cloning ··········································································27 - ii - Determination of DNA sequences from each sample ·········28 Analysis of SBV-specific DNA sequences ····························33 III Results and discussion ···································································35 Genome of SBVs belong to two different genotypes ·········35 Single nucleotide polymorphisms identification in analyzed sequences ························································································35 Phylogeny on SNP patterns in genotype 2100D0 ················41 IV Conclusion ·························································································43 Chapter Rapid detection of Israeli acute paralysis virus using multi-point ultra-rapid real-time PCR ·············································45 I Introduction ··························································································45 II Materials and methods ····································································47 Honeybee samples ·········································································47 Primer design ·················································································47 Construction of recombinant DNAs ·········································49 RNA extraction ··············································································51 Multi-point PCR ············································································51 Limit of detection ··········································································52 Assessment of multi-point UR-qPCR efficiency ·················53 Sequence analysis ·········································································53 - iii - III Results and discussion ···································································54 Comparison of single- and multi-point PCR ························54 Sequence analysis ·········································································59 Optimization of UR-qPCR ··························································61 IV Conclusion ·························································································66 Chapter Quantitative detection and evaluation of Melissococcus plutonius infection in honey bee ···························67 I Introduction ··························································································67 II Materials and methods ····································································68 Bacterial strain ···············································································68 Cultivation of M plutonius ························································68 Microscopic enumeration ·····························································70 Plate count ······················································································71 DNA extraction ··············································································72 PCR performance ···········································································72 Relationship between microscopic and PCR-based count ··73 Artificial infection of M plutonius to honeybee larvae ·····74 III Results and discussion ···································································75 Quantification of M plutonius by microscopy and plate - iv - count ································································································75 Molecular quantification of M plutonius using qPCR ·······76 Relationship between microscopic count and molecular count ································································································77 Artificial infection of M plutonius on honeybee larvae ···· 79 IV Conclusion ·························································································84 Chapter Evaluation of microscopic and molecular quantitative detection of Nosema ceranae in honeybees ···································86 I Introduction ··························································································86 II Materials and methods ····································································87 Sample preparation ········································································87 DNA extraction ··············································································89 Standard DNA and N ceranae -specific primers ·················89 Microscopic enumeration of Nosema spore ···························90 Molecular enumeration by quantitative PCR ·························90 Limit of Nosema detection by microscopic and molecular method ·····························································································91 Evaluation of N ceranae development in caged honeybees ········································································································93 III Results and discussion ···································································94 - v - Standard linear regression of UR-qPCR for quantification of N ceranae ················································································94 Quantification of N ceranae in caged bees fed by Bee happy solution ···············································································96 Impact of Bee happy solution on honeybee ···························98 Relationship between microscopic count and molecular count ······························································································100 Limit detection of N ceranae spores by microscopy ·······103 Limit detection of N ceranae using UR-qPCR ·················105 IV Conclusion ·······················································································106 Chapter Generation of monoclonal antibody for detection of N ceranae ······························································································107 I Introduction ························································································107 II Materials and methods ··································································108 Preparation of pure Nosema ceranae spore ························108 N ceranae confirmation by specific PCR ····························109 Antigen preparation ····································································110 Spore lysates ················································································110 Bradford assay ·············································································111 Mouse immunization ···································································111 Enzyme-linked Immunosorbent Assay (ELISA) ·················112 - vi - Myeloma cell culture ··································································114 Hybridoma generation ································································114 10 Selection of successful hybridoma cell line ·······················117 11 Production of monoclonal antibody ascites fluid ··············118 12 Western blotting ········································································119 13 Dot blotting ·················································································122 III Results and discussion ·································································123 Nesema ceranae confirmation from purified spore ···········123 Immunization of mice using N ceranae antigen ···············124 Selection of hybridoma for anti-N ceranae monoclonal antibody generation ·····································································125 Confirmation of anti-N ceranae monoclonal antibody ·····127 Production of monoclonal antibody in ascitic fluid ············128 IV Conclusion ·······················································································130 Part Molecular determination of adulterated honey ···············131 Chapter DNA identification of corn syrup adulterated honey ··················································································································131 I Introduction ························································································131 II Materials and method ····································································133 - 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183 - Appendix List of publications A Tai Truong , Byounghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, Seonmi Kim and Byoungsu Yoon 2019 Rapid detection of Israeli acute paralysis virus using multi-point ultra-rapid real-time PCR (UR-qPCR) Journal of Apicultural Research 58(5): 746-753 A Tai Truong , Jung Min Kim, Su Jin Lim, Mi Sun Yoo, Yun Sang Cho and Byoung Su Yoon 2018 Development of Ultra-rapid PCR System for Genotyping of Sacbrood Virus Journal of Apiculture 33(2): 83-98 A Tai Truong , Jung-Min Kim, Su-Jin Lim, Mi-Sun Yoo, Yun Sang Cho and Byoung-Su Yoon 2017 High Level of Sequence-Variation in Sacbrood Virus (SBV) from Apis mellifera Journal of Apiculture 32(4): 281-293 Somin Kim, Byounghee Kim, Moonjung Kim, Jungmin Kim, A Tai Truong and Byoungsu Yoon 2018 Detection of Sugar Beet (Beta vulgaris) - Specific Gene from Honey Made by Sugar of Sugar Beet Journal of Apiculture 33(3): 213-219 Byounghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, A Tai Truong and Byoungsu Yoon 2018 Detection of Sugar Cane (Saccharum officinarum)-specific Gene from Sugar and Sugar-honey Journal of Apiculture 33(3): 221-226 MoonJung Kim, JungMin Kim, ByeongHee Kim, SoMin Kim, A Tai Truong and ByoungSu Yoon 2018 Development of Nested Ultra-Rapid - 184 - PCR Method for the Accurate Detection of Acute Bee Paralysis Virus (ABPV) Journal of Apiculture 33(3): 165-180 Jung-Min Kim, A Tai Truong, So-Min Kim, Byoung-Hee Kim, Mun-Jung Kim and Byoung-Su Yoon 2018 Multi-Ultra-Rapid PCR against Viral Pathogens of Honeybee using Hive Debris Journal of Apiculture 33(3): 135-147 Byounghee Kim, Jungmin Kim, Somin Kim, Moonjung Kim, A Tai Truong , Kyoungjoo Cho & Byoungsu Yoon 2018 Detection of chronic bee paralysis virus using ultra-rapid PCR and nested ultra-rapid PCR Journal of Apicultural Research 58 (1):133-140 Jung-Min Kim, Su-Jin Lim, SoMin Kim, MoonJung Kim, ByoungHee Kim, A Tai Truong , Seonmi Kim, ByoungSu Yoon 2019 Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip Journal of Veterinary Science 20(6): e72 Jung-Min Kim, Su-Jin Lim, A Tai Truong , Ji-Hee Wang, Chil-Woo Lee1 and Byoung-Su Yoon 2017 Comparison between Specific DNA-amplifications using Recombinase Polymerase Amplification (RPA) and using Polymerase Chain Reaction (PCR) Journal of Apiculture 32(1): 41-50 Jung-Min Kim, Su-Jin Lim, A Tai Truong, Ki-Jeong Hong1 and Byoung-Su Yoon 2017 Development of Rapid Detection System for Small Hive Beetle (Aethina tumida) by using Ultra-Rapid PCR Journal of Apiculture 32(2): 119-131 - 185 - Somin Kim, Sujin Lim, Jungmin Kim, Byounghee Kim, A Tai Truong and Byoungsu Yoon 2017 Rapid Detection for Lysinibacillus fusiformis, a Suspicious Pathogen of Bombus terrestris, using Ultra-Rapid PCR Journal of Apiculture 32(3): 181-189 MoonJung Kim, Byoung-Hee Kim, SoMin Kim, A Tai Truong , Jung-Min Kim, Seonmi Kim and Byoung-Su Yoon 2019 Development of Ultra-rapid Nested PCR Method for Detection of Specific Gene of Tracheal Mite (Acarapis woodi) Journal of Apiculture 34(1): 15-26 Byounghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, A Tai Truong , Seonmi Kim and Byoungsu Yoon 2019 Quantitative Detection of Tropilaelaps in Hive by Specific Gene Detection from Hive Debris Journal of Apiculture 34(1): 27-37 Somin Kim, Byounghee Kim, Moonjung Kim, Jungmin Kim, A Tai Truong , Seonmi Kim and Byoungsu Yoon 2019 Development of Diagnostic System to Black Queen Cell Virus (BQCV) Using Multi-point Detection Journal of Apiculture 34(1): 39-46 - 186 - List of oral presentation A Tai Truong , Jung-Min Kim, Su-Jin Lim, Mi-Sun Yoo, Yun Sang Cho and ByoungSu Yoon 2017 Sacbrood virus (SBV) genotypes detection by Ultra-Rapid Real-time PCR The 32th Conference of the Apicultural Society of Korea A Tai Truong , Jung-Min Kim, Byoung-Hee Kim, So-Min Kim, Moon-Jung Kim, and Byoung-Su Yoon 2018 Accurate identification of Israeli acute paralysis virus by multi detection points using ultra-rapid real-time PCR The 33th Conference of the Apicultural Society of Korea A Tai Truong , Sedat Sevin, Jung-Min Kim, So-Min Kim, Moon-Jung Kim, and Byoung-Su Yoon 2018 Quantitative detection of Nosema ceranae using ultra-rapid real-time PCR and microscopic method The 34th Conference of the Apicultural Society of Korea A Tai Truong , Moonjung Kim, Byounghee Kim, Somin Kim, Jungmin Kim, Seonmi Kim and Byoungsu Yoon 2019 Amplification of Maize residual DNA in honey using ultra-rapid real-time PCR allows the detection of honey sample adulterated with various ratios of corn syrup sugar The 35th Conference of the Apicultural Society of Korea A Tai Truong , Moonjung Kim, Somin Kim, Seonmi Kim and Byoungsu Yoon 2019 Identification of seasonal honey by specific detection of typical plant compositions The 36th Conference of the Apicultural Society of Korea - 187 - ByoungSu Yoon, Moonjung Kim, Byoung-Hee Kim, So-Min Kim, Jung-Min Kim, A-Tai Truong 2019 Development of detection method as point-of-care using Ultra-rapid PCR and immunochromatography against 11 major pathogens in honeybee 46th Apimondia, international Apicultural congress, Quebec, Canada - 188 - List of poster presentation A Tai Truong , Jung-Min Kim, Su-Jin Lim, Mi-Sun Yoo, Yun Sang Cho and ByoungSu Yoon 2017 High level of sequence-variation in Sacbrood virus (SBV) from Apis mellifera The 32th Conference of the Apicultural Society of Korea A Tai Truong , Jung-Min Kim, Byoung-Hee Kim, So-Min Kim, Moon-Jung Kim, and Byoung-Su Yoon 2018 Degenerated PCR under short time of each step using ultra-rapid real-time PCR The 33th Conference of the Apicultural Society of Korea A Tai Truong , Jung-Min Kim, Byoung-Hee Kim, So-Min Kim, Moon-Jung Kim, and Byoung-Su Yoon 2018 Development of quantitative nested Ultra rapid real time PCR assay for genotyping Sacbrood virus from honeybee in Korea The 34th Conference of the Apicultural Society of Korea A Tai Truong , Byounghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, Seonmi Kim and Byoungsu Yoon 2019 Generation of monoclonal antibody for Nosema ceranae detection The 35th Conference of the Apicultural Society of Korea A Tai Truong , Somin Kim, Moonjung Kim, Seonmi Kim, Yoonkyu Lim and Byoungsu Yoon 2019 Direct amplification of residual DNA in honey for nectar source identification The 36th Conference of the Apicultural Society of Korea - 189 - 국문요지 박사학위논문 꿀벌 병원체의 정확한 검출법 및 사양꿀 판별을 위한 검사법 개발 경기대학교 대학원 생명과학과 트롱아타이 병원균은 전 세계적으로 꿀벌 군집 붕괴의 주요 요인이며, 꿀 생산성 저하의 원인이 된다 따라서 병원체의 정확하고 빠른 탐지를 위한 효과 적인 기술은 연구자들과 양봉가들 의 주요 관심사이다 이 연구의 목적은 바이러스 성 병 원체 (Sacbrood 바이러스 및 이스라엘 급성 마비 바이러스), 세균성 병원체 (Melissococcus plutonius) 및 곰팡이 병원체 (Nosema ceranae)의 진단을 위 한 분자 측정법을 개발하고 평가하는 것이다 5개의 SBV 유전자형의 빠른 검 출 및 정량적 측정을 위해 초고속 실시간 PCR (UR-qPCR) 시스템이 개발되 었다 10개의 꿀벌 샘플 A mellifera의 SBV 유전자형 분석은 4가지 샘플이 유전자형 SBVD0 및 SBVD51에 의해 감염된 것으로 나타났다 한편, 개의 샘플은 유전자형 SBVD0만을 갖고, 다른 4개의 샘플은 SBVD51만 가지므로 양성이었다 높은 서열 변이는 또한 SBV에서 밝혀졌으며, A mellifera의 4개 집단으로부터 유래된 16개의 SBV 서열에서 2개의 유전자형, SBVD51 및 SBVD0가 검출되었다 GenBank에서 가장 유사한 서열과 비교하여 총 87개의 불일치가 발견되었으며, 2100D0 유전자형에서 6개의 SNP 패턴과 2134D51 유 전자형에서 2개의 SNP 패턴이 SNP 위치를 기준으로 확인되었다 - 190 - 이스라엘 급성 마비 바이러스 (IAPV)는 미국에서 꿀벌의 군집 붕괴 장애 (CCD)에 관여하는 것으로 나타났습니다 그러나, 바이러스는 높은 수준의 유 전자 변이를 나타내고 일부 IAPV 균주는 관련 바이러스와 높은 정도의 상동 성을 나타내며, 감염된 꿀벌에서 IAPV의 검출은 비교적 어려운 작업이다 이 러한 장애물을 극복하기 위해 IAPV 게놈 내의 여러 검출 부위와 UR-qPCR 에 의존하는 새로운 분자 접근법이 개발되었다 새로운 시스템은 동시에 RNA-dependent RNA polymerase 유전자 (RdRp) 및 개의 capsid 유전자 (VP3 및 VP1)를 표적으로 삼았다 이 다분기점 PCR 접근법은 100 % IAPV 감염을 탐지 할 수 있는 능력을 가졌고, IAPV 감염의 86.96-95.6%만 탐지 할 수 있는 단일점 PCR을 능가하는 매우 효율적인 방법이었습니다 서열 분석은 RdRp가 2개의 캡시드 유전자보다 더 가변적이며, 제안된 방법의 특이성은 IAPV 및 Kashmir bee virus (KBV)에 의해 공동-감염된 샘플로부터 IAPV의 검출에 의해 입증되었다 유럽부저병(EFB)는 Melissococcus plutonius로 인한 세균성 질병입니다 감 염된 유충집단은 일반적으로 박테리아에 의해 죽고 악취가 납니다 효과적인 치료가 적용될 수 있는 감염 수준을 결정하기 위해서는 EFB 병원체의 정확한 진단을 위한 정량 분석이 중요합니다 UR-qPCR에 의한 검출법, 현미경계수, 평판계수 및 molecular count들이 연구에서 평가되었다 결과는 박테리아가 사 슬처럼 함께 부착되는 경향이 있기 때문에 평판계수 및 현미경계수가 M plutonius의 정확한 정량화가 힘들다는 것을 보여 주었다 분자적 계수 는 검 출된 M plutonius DNA copy 21개의 최소 한계를 나타내었고, DNA copy와 박테리아 수 사이의 관계가 확립되었다 또한, PCR 분석은 양봉장에서 EFB의 증상이 없는 감염된 유충의 이점을 보여주었다 유충에 대한 M plutonius의 인공 감염은 모두 동일한 양의 박테리아에 노출되었을 때 더 오래된 유충에서 유생의 높은 생존 및 발달 속도가 증가함을 보여 주었다 미포자 기생충 Nosema ceranae 는 꿀벌 개체군에서 세계적인 문제이며 벌 군집의 겨울 사망률을 높이는 것으로 알려졌다 이 연구는 감염된 성인 꿀벌 - 191 - 에서 N ceranae의 빠른 열거를 위해 초고속 실시간 PCR (UR-qPCR)을 이용 한 정량법을 개발했다 UR-qPCR 은 copy 의 N ceranae DNA 및 꿀벌 당 1.91 x 102 포자를 검출하는데 있어 현미경적 계수보다 더 민감했다 한편, 현 미경 검출은 검출 한계가 1.91 × 104 정제된 포자 / ml이고, 안정한 검출 수준 은 ≥1.91 × 105 포자 / ml였다 감염된 꿀벌로부터의 N ceranae 계산 결과, DNA 수는 포자 수보다 약 8.00 배 더 높았으며, N ceranae 에 의해 104-105 개의 DNA 수 / 꿀벌로 감염된 샘플은 현미경법으로는 검출하기 힘들었다 또 한, Nosema ceranae 의 면역학적 방법론 검출을 위해단클론항체를 제작하였 다 혼성세포 생산을 위해 마우스를 면역화시켜 정제된 포자를 사용하였다 mAb를 사용한 Western-blot의 결과는 mAb가 약 17kDa의 N ceranae 의 단 백질을 검출하는 것을 나타내었고, Dot blot를 통하여 은 × 103 포자에서 검 출 한계를 나타냈다 ELISA의 결과는 모든 희석율에서 복수를 사용한 N ceranae 검출 감도가 세포 배양 배지보다 훨씬 높았다 꿀은 꿀벌이 꽃에서 채취 한 천연의 달콤한 시럽으로, 인간의 삶에서 널리 알려진 맛과 건강상의 이점으로 인하여, 값 비싼 꿀은 경제적인 이유로 위조 의 표적이 되었다 본 연구에서는 옥수수 시럽 사양꿀 검출 및 단화꿀 판별을 위한 분자생물학적 방법이 개발되었다 정량적 검출법 결과는 20, 40, 60, 80% 의 옥수수 시럽과 섞인 꿀은 유전체 DNA양과 천연 꿀을 비교하여 검출 할 수 있음을 보여주었다 시판되는 7개의 꿀 샘플은 모두 옥수수 DNA가 천연꿀 수준만큼 낮다는 것이 검출됐다 단화꿀의 검출은 개화시기에 각 식물의 DNA 가 가장 많이 검출되어 다음 달의 샘플에서 남아있음을 보여주었다 식물 조 성은 5월에서 7월까지 증가된 꿀 샘플에 존재했다 그러나 각 식물 종의 DNA 양은 감소하는 경향이 있었다 시판 단화꿀의 확증적인 결과는 벚꽃 꿀만이 많은 양의벚나무 특이 유전자를 가지고 있다는 것이었다 다른 단화 꿀들은 예상된 DNA 보다 적은 양의 DNA를 가지고 있기 때문에 시정되어야 할 것 이다 - 192 - - 193 - .. .Development of methods for accurate detection of honeybee pathogens and molecular determination of adulterated honey A dissertation submitted to the faculty of Graduate School of Kyonggi... collapse of honeybee colony worldwide, and cause of decrease of honey productivity Therefore, the efficient technologies for accurate and rapid detection of the pathogens are the major concern of researchers... developed for the fast detection and quantitative determination of each SBV genotype in infected honeybee from this study Designation of specific primer-pairs and construction of standard DNAs for