Development of Ultra-rapid PCR Method for Detection of Specific Genes of Honeybee Pathogens and Molecular Test Method for Plant Genes Present in Adulterated Honey

다국어 초록 (Multilingual Abstract)

This study aimed to develop a rapid and accurate detection method that can overcome the limitations of conventional PCR by applying the ultra-rapid nested PCR method and ultra-rapid 3-points PCR method. Efficient expression and purification of kSBV-RdRp-specific proteins and Anti-kSBV-RdRp monoclonal antibodies were evaluated. I attempted to construct a hybridoma cell line for specific antibodies. In addition, I attempted to develop a rapid and sensitive genetic test for distinguishing adulterated honey from genes remaining in honey.


  The rapid and sensitive honeybee pathogens detection method using ultra-rapid PCR was developed. In order to increase specificity, definitive tests using nested PCR method and 3-points PCR method were developed. In the nested PCR method, the amplification products of the primary PCR were reconfirmed by the secondary PCR. The reliability of the results could be improved. 3-points PCR confirms specific amplification of three different gene sites in one pathogen. It is designed to make more accurate diagnosis. The proposed detection method enables on-site detection with the rapidity of ultra-rapid real-time quantitative PCR. The quantitative, specificity and accuracy of the nested PCR method and 3-points PCR methods are expected to be useful as a definitive test for overall pathogens.


  The kSBV-RdRp specific recombinant gene and expression system of E. coli were used to over-express kSBV-RdRp specific protein. Specific proteins were purified using MBP agarose resin. Using the purified protein as an antigen, a cell line was produced to generate Anti-kSBV-RdRp monoclonal antibody. Cell fusion results in a hybridoma cell line that generates monoclonal antibodies against kSBV-RdRp specific antigens. In the future, the monoclonal antibody is expected to be used not only for the detection and quantification of kSBV-RdRp specific proteins but also for the production of kSBV-RdRp specific recombinant antibodies.


  Efforts to distinguish between natural honey and adulterated honey are ongoing. However, there is a problem that it is difficult to distinguish the adulterated honey mixed with natural honey or made of sugarcane/sugarbeet-sugar. This study demonstrates the presence of specific genes of sugarcane and nectar-source plant remaining in natural or adulterated honey. It is hoped that the molecular test method using this unique gene can be developed into a new adulterated honey discrimination method.

국문 초록 (Abstract)

본 연구는 초고속 nested PCR 및 3-points PCR을 적용시킴으로써 conventional PCR의 한계를 극복할 수 있는 신속하고 정확한 검출법을 개발하고자 하였다. kSBV-RdRp 특이 단백질의 효과적인 발현과 정제, 이를 이용한 Anti-kSBV-RdRp 단일클론항체를 평가하여, 특이 항체에 대한 hybridoma cell line을 구축하고자 하였다. 또한 꿀에 잔류하는 유전자로부터 사양꿀 판별을 위한 신속하고 민감한 유전자 검사법을 개발하고자 하였다.


초고속 PCR을 사용하여 신속하고 민감한 꿀벌 병원체 검출법을 개발하고자 하였으며, 특이성을 높이기 위하여 nested PCR 및 3-points PCR을 이용한 확정검사를 개발하였다. Nested PCR법은 1차 PCR의 증폭산물이 2차 PCR에 의해 재검됨으로써, 결과해석의 신뢰도를 높일 수 있었다. 3-points PCR법은 1개 병원체에서 서로 다른 3개 유전자 부위에 대한 특이 증폭을 확인하는 것으로, 보다 정확한 진단이 가능하도록 고안하였다. 제안하는 검사법은 초고속 실시간 PCR의 신속성으로 현장검사를 가능하게 하였으며, 2차 검사인 nested PCR법 및 3-points PCR법의 높은 정량성과 특이성, 정확성은 병원체 전반을 대상으로 하는 확정 검사법으로 유용하게 사용될 것으로 기대한다.


kSBV-RdRp 특이 재조합 유전자와 대장균 발현 시스템을 이용하여 kSBV-RdRp specific protein을 과발현하고, MBP agarose resin을 이용하여 특이 단백질을 정제하였다. 정제된 단백질을 항원으로, Anti-kSBV-RdRp 단일클론항체를 생산하는 cell line을 제작하였다. Fusion 결과 kSBV-RdRp 특이 항원에 대한 서로 다른 단일 클론항체를 생산하는 6개의 서로 다른 hybridoma cell line을 구축할 수 있었으며, 추후 본 단일클론 항체는 kSBV-RdRp specific protein에 대한 검출 및 정량에 사용될 뿐만 아니라 kSBV-RdRp 특이 재조합 항체를 제작하는데 이용될 것으로 기대된다.


사양꿀과 천연꿀의 구분을 위한 노력이 계속되고 있으나, 사양꿀과 천연꿀이 혼합되거나 사탕무 설탕으로 제조된 사양꿀의 경우 구분이 어렵다는 문제점이 있다. 본 연구는 사양꿀 및 천연꿀에 잔류하는 초미량의 사탕수수 및 밀원수의 고유 유전자의 존재를 입증하고, 이 고유 유전자를 이용한 유전자 검사법을 새로운 사양꿀 판별법으로 발전할 수 있기를 기대한다.

목차 (Table of Contents)

• Part Ⅰ. Development of Ultra-rapid PCR Method for Detection of Specific Gene of Honeybee Pathogens 1
• Ⅰ. Introduction 1
• 1. The pathogen of honeybee 1
• 1) Viral pathogen 1
• 2) Parasite 4
• 2. Ultra-rapid real-time quantitative PCR (URRT-qPCR) 6
• 3. Monoclonal antibody 10
• 4. Purpose of this study 13
• Ⅱ. Materials and Methods 15
• 1. Materials 15
• 1) Bacterial strains 15
• 2) Culture medium 16
• (1) Luria-Betani medium 16
• (2) Agar plate 17
• (3) Psi broth 18
• (4) SOC medium 19
• 3) Plasmid vector 20
• (1) pBlueXcm vector 20
• (2) pTOP vector 22
• (3) pGEM vector 24
• (4) pMAL-c2 vector 27
• 4) Composition of reagents for purification 29
• 5) Composition of reagents for SDS-PAGE 30
• 6) Composition of reagents for ELISA test 33
• 7) Composition of reagents for cell culture 35
• 2. Methods 37
• 1) Collection of samples 37
• 2) Gene extraction and Artificial synthesis 38
• (1) Total RNA extraction 38
• (2) Reverse transcription 39
• (3) DNA extraction 40
• ⅰ. DNeasy Blood & Tissue Kit 40
• ⅱ. Rapid freezing method (using LN2) 41
• ⅲ. PathoGene-spin DNA/RNA Extraction Kit 42
• (4) plasmid DNA isolation 43
• (5) Artificial synthesis 44
• ⅰ. Artificial synthesis of ABPV-specific gene 44
• ⅱ. Artificial synthesis of A. woodi-specific gene 48
• 3) Molecular cloning 50
• (1) Polymerase chain reaction (PCR) 50
• (2) Restriction of plasmid DNA 51
• (3) Electrophoresis 52
• (4) Ligation 53
• ⅰ. Conventional method 53
• ⅱ. pTOPcloner TA core Kit 55
• ⅲ. pGEM-T and pGEM-T Easy Vector System Kit 55
• (5) Making competent cell 56
• (6) Transformation 58
• (7) Screening and Sequencing 59
• ⅰ. Colony PCR 59
• ⅱ. Restriction screening 61
• ⅲ. Sequencing 61
• 4) Design and evaluation of specific primer 62
• (1) Design and selection of specific primer 62
• ⅰ. Design of ABPV-specific primer 62
• ⅱ. Design of A. woodi-specific primer 62
• ⅲ. Design and selection of DWV-specific primer 63
• ⅳ. Design and selection of KBV-specific primer 63
• (2) Establish optimal annealing temperature of specific primer 67
• (3) Measurement of detection limit 68
• 5) Detection using URRT-qPCR 69
• 6) Over-expression and Purification 70
• 7) SDS-PAGE 71
• 8) Bradford assay 73
• 9) ELISA test 74
• 10) Cell fusion 75
• (1) Sp2/0 cell thawing 75
• (2) Cell culture 76
• (3) Injection 77
• (2) Cell fusion 78
• (3) Freezing 80
• Ⅲ. Results and Discussion 81
• 1. Artificial synthesis of specific gene 81
• 1) Artificial synthesis of ABPV-specific gene 81
• 2) Artificial synthesis of A. woodi-specific gene 82
• 2. Confirmation of optimal annealing temperature of each specific gene at URRT-qPCR 83
• 1) Optimal condition of ABPV-specific PCR 83
• 2) Optimal condition of A. woodi-specific PCR 86
• 3) Optimal condition of DWV-specific PCR 88
• 4) Optimal condition of KBV-specific PCR 90
• 3. Confirmation of detection limit 93
• 1) Detection limit of ABPV-specific PCR 93
• 2) Detection limit of A. woodi-specific PCR 102
• 3) Detection limit of DWV-specific PCR 105
• 4) Detection limit of KBV-specific PCR 108
• 4. Application with infected samples 113
• 1) Detection of ABPV-specific gene 113
• 2) Detection of A. woodi-specific gene 119
• 3) Detection of DWV-specific gene 125
• 4) Detection of KBV-specific gene 133
• 5. Purification and quantity of kSBV-RdRp 138
• 6. Selection of Anti-kSBV-RdRp specific hybridoma 141
• Ⅳ. Conclusions 143
• Part Ⅱ. Development of Genetic Test for Confirmation of Adulterated Honey 147
• Ⅰ. Introduction 147
• 1. Natural honey and Adulterated honey 146
• 2. Importance of adulterated honey distinction method 149
• 3. Usefulness of genetic test method 150
• 4. Purpose of this study 152
• Ⅱ. Materials and Methods 153
• 1. Materials 153
• 1) Binding buffer 153
• 2) Washing buffer 153
• 3) CTAB/NaCl solution 154
• 2. Methods 155
• 1) Collection of samples 155
• (1) Chestnut (Castanea crenata) and Locust (Robisia psuedoacacia) 155
• (2) Sugarcane (S. officinarum) 155
• (3) Honey 155
• 2) DNA extraction 157
• (1) DNA extraction from plant 157
• (2) DNA extraction using CTAB method 158
• (3) DNA extraction from honey 159
• (4) Plasmid DNA isolation 159
• 3) Molecular cloning 160
• (1) pTOPcloner TA core Kit 160
• (2) pGEM-T and pGEM-T Easy Vector System Kit 160
• 4) Design and evaluation of specific primer 161
• (1) Design and selection of specific primer 161
• ⅰ. Design of chestnut-specific primer 161
• ⅱ. Design of locust-specific primer 161
• ⅲ. Design and selection of sugarcane-specific primer 162
• (2) Establish optimal annealing temperature of specific primer 166
• (3) Measurement of detection limit 167
• 5) Rapid detection using URRT-qPCR 168
• Ⅲ. Results and Discussion 169
• 1. Confirmation of optimal annealing temperature of each specific gene at URRT-qPCR 169
• 1) Optimal condition of chestnut-specific PCR 169
• 2) Optimal condition of locust-specific PCR 172
• 3) Optimal condition of sugarcane-specific PCR 173
• 2. Confirmation of detection limit 176
• 1) Detection limit of chestnut-specific PCR 176
• 2) Detection limit of locust-specific PCR 179
• 3) Detection limit of sugarcane-specific PCR 182
• 3. Application and quantity from honey 185
• 1) Detection of chestnut-specific gene from honey 185
• 2) Detection of locust-specific gene from honey 190
• 3) Detection of sugarcane-specific gene from honey 195
• Ⅳ. Conclusions 201
• References 203
• Appendix 211
• Abstract in Korean (국문요지) 215