수목에 발생하는 phytoplasma의 분자적 특성 및 고리매개등온증폭법을 이용한 신속 진단 = Molecular characterization of phytoplasmas infecting woody plants and rapid diagnosis using loop-mediated isothermal amplification

다국어 초록 (Multilingual Abstract)

Phytoplasma infections in Korea have been increasingly reported across various woody plants, including fruit trees, forest trees, urban forests, and ornamental trees. However, molecular information based on secA and rp operon sequences remains limited for many hosts, restricting comparative analyses of host associated genetic diversity.
Diagnosis is further complicated by extremely low pathogen titers, uneven distribution within plant tissues, and the frequent absence of clear symptoms during early infection stages. Conventional PCR based methods require time consuming procedures and pose a high risk of cross contamination, limiting their applicability for rapid field diagnostics.
Loop-mediated isothermal amplification(LAMP) has emerged as a practical alternative, allowing nucleic acid amplification at 60-65 ℃ using a thermostable polymerase and a set of multiple primers, and enabling rapid detection through simple visual inspection or fluorescence based readout without electrophoresis.
To improve phytoplasma diagnostics and genetic characterization, phytoplasmas infecting woody hosts in Korea were analyzed using secA and rp operon sequences to determine their phylogenetic relationships across 16Sr groups and subgroups. Based on these molecular data, a tuf gene based LAMP diagnostic assay was developed to simultaneously detect three 16Sr groups occurring in Korean woody plants, providing a rapid, accurate, and field applicable tool for phytoplasma identification.

목차 (Table of Contents)

• Ⅰ. 서론 1
• Ⅱ. 재료 및 방법 4
• 1. 대상 시료 및 보관 4
• 2. Total DNA 추출 7
• 3. PCR 증폭 9
• 4. RFLP 분석 15
• 5. 염기서열 분석 및 계통분석 17
• 6. LAMP 진단법 18
• 가. LAMP 반응 및 검출 방법 18
• 나. LAMP 반응의 검출한계 분석 23
• Ⅲ. 결 과 25
• 1. Phytoplasma 감염 수목의 증상 관찰 25
• 2. PCR 분석을 통한 phytoplasma 검출 결과 30
• 3. RFLP 분석 결과 32
• 4. 염기서열 및 계통분석 결과 36
• 가. rp operon 유전자 분석 36
• 나. secA gene 유전자 분석 39
• 5. LAMP 진단법 결과 43
• 가. End-point LAMP 반응 결과 43
• 나. Eye-detection LAMP 반응 결과 46
• 다. 검출한계(LOD) 분석 결과 49
• Ⅳ. 고찰 51
• Ⅴ. 결론 54
• 참고문헌 55