콩 우량종자 공급을 위한 대량시료 바이러스 검사법 개발 = Development of Large-Scale Sample Virus Detection Methods for Supplying High-Quality Soybean Seeds

다국어 초록 (Multilingual Abstract)

A simple real-time RT-PCR based assay was developed to investigate virus infection rates in large-scale soybean seed lots. Conventional qualitative RT-PCR requires numerous assays of individual samples within a population, whereas the method developed in this study enabled estimation of infection rates at the population level with a single real-time RT-PCR analysis. To validate the method, seeds of three government-released soybean cultivars (‘Seonpung’, ‘Daechan’, and ‘Daewonkong’) were tested for soybean yellow mottle mosaic virus (SYMMV) infection using RT-PCR, RT-PCR followed by nested PCR, and real-time RT-PCR. Detection of low-level SYMMV infections was difficult using RT-PCR alone; however, both RT-PCR followed by nested PCR and real-time RT-PCR successfully identified low-level infected seeds, and their results were consistent. In particular, real-time RT-PCR enabled quantification of target gene copy numbers in individual infected seeds, and cultivar-specific mean copy numbers were established. Using these values, population-level infection rates could be estimated through a single assay, which closely matched the results of qualitative testing of individual seeds. The large-scale quantitative virus assay developed in this study is expected to be highly useful not only for quality control in soybean seed production but also for the diagnosis of regulated non-quarantine pests with defined tolerance thresholds.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 3
• 1. 공시 시료 3
• 2. 소집단 total RNA 추출 6
• 3. 진단용 프라이머 및 프로브 7
• 4. 소집단 정량분석 9
• 5. 개별 종자 total RNA 추출 9
• 6. 개별 종자 정성 및 정량 진단 9
• 6.1. 개별 종자 RT-PCR 9
• 6.2. 개별 종자 nested PCR 10
• 6.3. 개별 종자 real-time RT-PCR 10
• 7. 감염률 추정 10
• 8. 통계 분석 11
• 8.1. 정성 및 정량 분석간 일치도 분석 11
• 8.2. Ct 임계치 교차 검증 11
• 8.3. 표본 수 산정 12
• Ⅲ. 결 과 13
• 1. 소집단 정량 진단 13
• 2. SYMMV 감염률 추정 17
• 2.1. SYMMV 정성 및 정량 진단 17
• 2.1.1. 개별 종자 SYMMV 정성 진단 17
• 2.1.2. 개별 종자 SYMMV 정량 진단 22
• 2.2. 품종별 SYMMV 평균 복제량 결정 27
• 2.3. 품종별 SYMMV 감염률 산출 29
• 2.4. 통계 분석 33
• 2.4.1. 정성 및 정량 분석간 일치도 분석 33
• 2.4.2. Ct 임계치 교차 검증 34
• 3. SMV 감염률 추정 35
• 3.1. SMV 정성 및 정량 진단 35
• 3.1.1. 개별 종자 SMV 정성 진단 35
• 3.1.2. 개별 종자 SMV 정량 진단 37
• 3.2. 품종별 SMV 감염률 산출 39
• Ⅳ. 고 찰 40
• Ⅴ. 적 요 42
• Ⅵ. 참 고 문 헌 43
• Ⅵ. 영 문 초 록 47