种传西瓜细菌性果斑病菌快速分子检测方法研究
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摘要
由燕麦嗜酸菌西瓜亚种（Acidovorax avenae subsp.citrulli）引起的西瓜细菌性果斑病是西瓜等葫芦科作物的典型种传病害，也是重要的检疫对象之一。由于化学药剂处理无法完全清除该病原，因此，种子检疫成为控制该病的重要手段。由于西瓜种子较大，带菌率偏低，传统方法常导致微量病原的漏检。因此，迫切需要开发简单易行、快速灵敏的检测方法。本文研究了几种分子生物学方法对种传西瓜细菌性果斑病菌的检测，主要结果如下：
1.采用直接高温裂解释放DNA，进行PCR扩增，无需专门DNA提取，以检测灵敏度、稳定性、时间及成本为指标对Bio-PCR、MF-PCR、IMS-PCR、IC-PCR、Direct-PCR、DAS-ELISA6种方法进行了综合评价的结果表明：IC-PCR和IMS-PCR检测灵敏度和重复性最高，最低检测限分别为4.7×10~2cfu/mL和4.7×10~2~4.7×10~3cfu/mL，0.1%带菌率的种子阳性检出率66.7%，检测时间比Direct-PCR的4h和MF-PCR的5h要长，但仍可控制在12h内。
2.建立以BX-L1/BX-R5为外侧引物，BX-L1/BX-S-R2为内侧引物的Nested-PCR检测方法，优化50μL反应体系：7.5μL10×PCR buffer（25mM MgCl2）、4μLdNTP（D4030RA，2.5mM）、引物（5μM/L）各3μL，0.25μLTaq DNA酶（DR001B，5U/μL）。该体系下以直接高温裂解释放DNA进行PCR扩增的结果清晰。
3.采用Nested-PCR方法，以50μL优化体系对Aac菌悬液、模拟带菌种子提取液和不同带菌率的种子洗液进行检测，结果表明：最低检测限2.4×10~1cfu/mL，0.1%带菌率的种子阳性检出率100%。
4.建立了可视化抗体芯片检测方法。以硝酸纤维素膜为基片，以西瓜细菌性果斑病菌为模式病原，对其特异性捕获抗体以阵列模式点样排布固定制成抗体阵列，并对点样浓度、封闭条件、杂交反应条件、显色时间等主要参数进行优化。
5.采用可视化抗体芯片检测技术对Aac菌悬液、模拟带菌种子提取液和不同带菌率的种子洗液进行检测的结果表明：最低检测限2.4×10~4~2.4×10~5cfu/mL，以1/25的捕获抗体用量，以及1/8的检测时间便可达到ELISA的同等检测能力，且成本低，操作简便。
Bacterial fruit blotch of watermelon caused by Acidovorax avenae subsp. citrulli (Aac) isone of important national plant quarantine disease in China. Aac mainly infects watermelonsor other melons which belong to Cucurbitaceous crops. It has become the most seriousdiseases threatening the watermelon production and it can incite devastating disease epidemicswith the favorable environmental conditions. Contaminated watermelon seed is the primarysource of the disease spreading. Until now,the most efficient disease-management strategy isto eliminate the infected seeds prior the planting. However, the relatively low proportion ofinfected-seed makes the disease management especially difficult. Other traditional detectionmethods require extra time, labor intensive but also need the knowledge of the pathogeniccharacteristic and the disease symptoms. Therefore, it is necessary to develop accurate, rapidand sensitive methods for detecting Aac invasion and preventing of the spread of disease.
The objective of this study was to explore and develop molecular detection techniques forAac.The results showed as below:
1. A series dilution of bacterial suspension and seed extracts suspension were boiled torelease DNA for the Bio-PCR,MF-PCR,IMS-PCR,IC-PCR,Direct-PCR and DAS-ELISAanalysis. Comprehensive comparisons were made based on the sensitivity, stability, time andcost. The data showed that both IC-PCR and IMS-PCR are considerable higher sensitivitymethods, the sensitivity of IC-PCR could reach4.7×10~2cfu/mL and that of IMS could reach4.7×10~2cfu/mL~4.7×10~3cfu/mL. Meanwhile, the true positive detection rate was66.7%in0.1%rate of infected-seed. Although the procedure time was longer than Direct-PCR (4h) andMF-PCR(5h), it finished within12h. The expenses of the detection those two methods wasalso very low. Therefore, our results suggested that IC-PCR and IMS-PCR are sensitive,specific, rapid, reproducible, and economical methods for detecting Aac in watermelon seeds.
2. The specific primer set BX-L1/BX-R5and BX-L1/BX-S-R2derived from BOXPCRwere selected to develop a nested PCR in single assay of Aac. The optimal PCR reactionsystem was50μL:7.5μL10PCR buffe（r25mM MgCl2）,4μL dNTP（D4030RA，2.5mM）,3μL primer(5μM/L），0.25μL Taq（DR001B,5U/μL）. the direct pyrolysis was used to releaseDNA as template in this system, the amplified result was clear.
3. The DNA of Aac suspension, mocked seeds, and different carrier rate of seeds wereamplified by50μL system of nested-PCR, it was indicated that the lowest detection limit canreach2.4×10~1cfu/mL, and the positive detection rate was100%in0.1%of infected-seed.
4. Setting cellulose nitrate film as vector and bacterial fruit blotch of watermelon as amodel pathogen, the specificity of the capture antibody was arrayed as an array pattern samplefor arrangement and fixation; we established an antibody microarray detection method. Themain parameters of sample concentration, incubation condition, hybridization reactionconditions, chromogenic time were optimized, and a visible protein chip was established.
5. Using the visible antibody microarray detection technology, the suspension of Aac,mocked seeds, and infected-seed with varies infection were analyzed. My results showed that:the lowest detection capability reached2.4×10~4cfu/mL~2.4×10~5cfu/mL. In the proteinchip system, only1/25dosage of the capture antibody and1/8of the detection time couldreach the same detection capability that achieved by ELISA. In addition, the cost of detectionwas low and the procedure was simple and convenient.
1.采用直接高温裂解释放DNA，进行PCR扩增，无需专门DNA提取，以检测灵敏度、稳定性、时间及成本为指标对Bio-PCR、MF-PCR、IMS-PCR、IC-PCR、Direct-PCR、DAS-ELISA6种方法进行了综合评价的结果表明：IC-PCR和IMS-PCR检测灵敏度和重复性最高，最低检测限分别为4.7×10~2cfu/mL和4.7×10~2~4.7×10~3cfu/mL，0.1%带菌率的种子阳性检出率66.7%，检测时间比Direct-PCR的4h和MF-PCR的5h要长，但仍可控制在12h内。
2.建立以BX-L1/BX-R5为外侧引物，BX-L1/BX-S-R2为内侧引物的Nested-PCR检测方法，优化50μL反应体系：7.5μL10×PCR buffer（25mM MgCl2）、4μLdNTP（D4030RA，2.5mM）、引物（5μM/L）各3μL，0.25μLTaq DNA酶（DR001B，5U/μL）。该体系下以直接高温裂解释放DNA进行PCR扩增的结果清晰。
3.采用Nested-PCR方法，以50μL优化体系对Aac菌悬液、模拟带菌种子提取液和不同带菌率的种子洗液进行检测，结果表明：最低检测限2.4×10~1cfu/mL，0.1%带菌率的种子阳性检出率100%。
4.建立了可视化抗体芯片检测方法。以硝酸纤维素膜为基片，以西瓜细菌性果斑病菌为模式病原，对其特异性捕获抗体以阵列模式点样排布固定制成抗体阵列，并对点样浓度、封闭条件、杂交反应条件、显色时间等主要参数进行优化。
5.采用可视化抗体芯片检测技术对Aac菌悬液、模拟带菌种子提取液和不同带菌率的种子洗液进行检测的结果表明：最低检测限2.4×10~4~2.4×10~5cfu/mL，以1/25的捕获抗体用量，以及1/8的检测时间便可达到ELISA的同等检测能力，且成本低，操作简便。
Bacterial fruit blotch of watermelon caused by Acidovorax avenae subsp. citrulli (Aac) isone of important national plant quarantine disease in China. Aac mainly infects watermelonsor other melons which belong to Cucurbitaceous crops. It has become the most seriousdiseases threatening the watermelon production and it can incite devastating disease epidemicswith the favorable environmental conditions. Contaminated watermelon seed is the primarysource of the disease spreading. Until now,the most efficient disease-management strategy isto eliminate the infected seeds prior the planting. However, the relatively low proportion ofinfected-seed makes the disease management especially difficult. Other traditional detectionmethods require extra time, labor intensive but also need the knowledge of the pathogeniccharacteristic and the disease symptoms. Therefore, it is necessary to develop accurate, rapidand sensitive methods for detecting Aac invasion and preventing of the spread of disease.
The objective of this study was to explore and develop molecular detection techniques forAac.The results showed as below:
1. A series dilution of bacterial suspension and seed extracts suspension were boiled torelease DNA for the Bio-PCR,MF-PCR,IMS-PCR,IC-PCR,Direct-PCR and DAS-ELISAanalysis. Comprehensive comparisons were made based on the sensitivity, stability, time andcost. The data showed that both IC-PCR and IMS-PCR are considerable higher sensitivitymethods, the sensitivity of IC-PCR could reach4.7×10~2cfu/mL and that of IMS could reach4.7×10~2cfu/mL~4.7×10~3cfu/mL. Meanwhile, the true positive detection rate was66.7%in0.1%rate of infected-seed. Although the procedure time was longer than Direct-PCR (4h) andMF-PCR(5h), it finished within12h. The expenses of the detection those two methods wasalso very low. Therefore, our results suggested that IC-PCR and IMS-PCR are sensitive,specific, rapid, reproducible, and economical methods for detecting Aac in watermelon seeds.
2. The specific primer set BX-L1/BX-R5and BX-L1/BX-S-R2derived from BOXPCRwere selected to develop a nested PCR in single assay of Aac. The optimal PCR reactionsystem was50μL:7.5μL10PCR buffe（r25mM MgCl2）,4μL dNTP（D4030RA，2.5mM）,3μL primer(5μM/L），0.25μL Taq（DR001B,5U/μL）. the direct pyrolysis was used to releaseDNA as template in this system, the amplified result was clear.
3. The DNA of Aac suspension, mocked seeds, and different carrier rate of seeds wereamplified by50μL system of nested-PCR, it was indicated that the lowest detection limit canreach2.4×10~1cfu/mL, and the positive detection rate was100%in0.1%of infected-seed.
4. Setting cellulose nitrate film as vector and bacterial fruit blotch of watermelon as amodel pathogen, the specificity of the capture antibody was arrayed as an array pattern samplefor arrangement and fixation; we established an antibody microarray detection method. Themain parameters of sample concentration, incubation condition, hybridization reactionconditions, chromogenic time were optimized, and a visible protein chip was established.
5. Using the visible antibody microarray detection technology, the suspension of Aac,mocked seeds, and infected-seed with varies infection were analyzed. My results showed that:the lowest detection capability reached2.4×10~4cfu/mL~2.4×10~5cfu/mL. In the proteinchip system, only1/25dosage of the capture antibody and1/8of the detection time couldreach the same detection capability that achieved by ELISA. In addition, the cost of detectionwas low and the procedure was simple and convenient.
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