柑桔黄龙病菌特异性基因片段克隆及检验检疫技术研究
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摘要
柑桔黄龙病(Citrus Huanglongbing Disease,HLB)由韧皮部难养菌引起,是严重影响全世界柑桔生产的重大检疫性病害之一。柑桔发病后轻者影响产量和品质,重者造成柑桔树的枯死。目前除严格防治木虱和挖除病树外,国内外对柑桔黄龙病尚无很好的根除方法,唯有通过设立和建设无病区、加强植物检疫严防病害传入。因此,加强病害的早期诊断和检测技术的研究尤为重要。柑桔黄龙病菌的传统检测技术都存在一定的局限性,如特异性差、灵敏度低,尤为关键的是不能对病害进行早期快速准确检测。而核酸水平的分子检测技术具有快速、特异、灵敏的特点,可以满足国内外现代植物检疫的要求。
研究目的:本文对柑桔黄龙病菌核酸分子检测技术和生物大分子常温贮藏技术进行了系统的研究,旨在建立起快速准确稳定安全的柑桔黄龙病菌分子检测技术体系,实现柑桔样品的快速检验,为柑桔非疫区生产建设和病害预警监测提供一种分子检测技术和实用工具。
研究方法:利用CTAB 法,试剂盒法和浸泡过滤法提取柑桔黄龙病菌的核酸DNA;利用引物和探针设计软件Primer Premier 5.0 进行引物和探针的设计;PCR同步扩增柑桔叶面腐生菌,柑桔溃疡病菌和衰退病菌等等植物细菌和病原物来验证检测的特异性;PCR 扩增含有靶片段的质粒DNA 测定检测的灵敏度;通过在不同DNA 扩增仪和温度控制方式下的扩增程序优化测定检测的稳定性。将靶片段与克隆载体pMD-18T 连接后,用CaCl2法转化大肠杆菌(E. coli,JM109),然后通过测序来验证PCR 扩增产物的准确性;以重组质粒和转化的大肠杆菌构建了无害化阳性参照体系。采用预混PCR 反应液中加入生物活性分子稳定剂并冷冻干燥固相化处理的方法研制出能常温贮存的方便有效的检测试剂盒。利用优化的分子检测体系对江西、浙江、广西、湖南、重庆和四川等地采集的柑桔样品进行了PCR实际检测。利用iCycler iQ定量PCR 仪对柑桔黄龙病的定量检测做了初步的研究。
研究结果:
1) 通过比较研究不同制样方法对检测效果的影响,确定浸泡过滤法能有效地排除植物色素、蛋白质、多糖等物质对PCR 反应的干扰,制备适合于PCR 反应的靶细菌DNA,避免PCR 反应的假阴性发生。
2) 根据柑桔黄龙病菌核糖体蛋白基因设计筛选了不同的引物对,筛选出特异性强稳定性好灵敏度高的引物对CQULAF3/CQULAR3,并由此建立了准确、快速、灵敏、稳定、安全的柑桔黄龙病菌PCR 检测体系。
3) 在引物CQULAF3/CQULAR3 扩增产物中设计了引物CQULAF4/CQULAR4,
Citrus Huanglongbing Disease (HLB), caused by an uncultured phloem-restricted, G-bacterium, is one of severe quarantine diseases domestically and abroad. After the citrus trees are infected, the yield and quality are affected, even the whole is dead. At present the appropriate method is to eradicate and burn infected plant material. In order to prevent the disease from spreading in citrus plant area of China we should develop disease-free area of citrus production. Therefor it is important to do research on detection technique. There are some disadvantages i.e. low specificity and sensitivity in the routine detection techniques. It is hard to meet the Quaratine requirement and conduct presentation diagnosis of HLB. The detection according to nucleic acid molecule is rapid, sensitive and special and can meet modern plant quarantine requirement domestically and abroad.
Objective: This thesis aims at developing detection system of DNA molecule with specificity, rapidness, accuracy and stabilization and providing practical tool of disease diagnosis for establishment of disease-free area and precautionary monitoring of HLB through the research on the molecule detection techniques of HLB and diagnosis kit stored at room temperature systemically.
Methods: First of all, DNA of Candidatus Liberibacter asiaticus was extracted by the methods of CTAB,reagent kits and soaking-filtration. Primer pairs and a probe were designed using software Primer Premier 5.0. The specificity was validated through PCR detection of the saprophytic microorganism of citrus leaf surface, Tristeza virus and bacteria of Xac etc plant pathogens or G-/ G+ bacteria. The sensitivity of PCR was determinated by amplification of the bacteria DNA and target sequence inserted in recombinant plasmid solution with ten-fold serial dilution. PCR profiles were adjusted by using different type of Thermocycler in different temperature control model, which ensured the stability of PCR detection. After being inserted into clone vector and recombinant plasmid transformed into the E. coli strain (JM109), the target DNA fragment was sequenced, and amplification fidelity of PCR was verified by sequences alignment using BLASTn. Then a harmless positve reference system was constructed with recombinant plasmid DNA. Solidifying PCR reagent kit was produced by adding macromolecular stabilizer (MS) into PCR mixed reagents and vacuum freeze-drying treatment. Field samples with or without disease symptom of citrus collected from
Guangxi, Zhejiang, Jiangxi, Sichuan provinces and Chongqing area were detected by PCR assay. The primary study on quantitative detection of HLB pathogen (Ca.L.asiaticus) was made by the iCycleriQ Realtime detection system. Experiment Results: ⑴DNA of Ca.L.asiaticus extraction methods were compared. Among them the soaking-filtration is the best one, which remove plant pigment, protein and polysaccharide totally, pure target DNA was obtained and false negativity of PCR reaction was avoided effectively. ⑵Specific primers (CQULAF3/LAR3) which have good specificity, sensitivity and stability for HLB detection, were designed based on published sequence encoding a ribosome protein gene and detection system of DNA molecule with rapidness, accuracy, stabilization have been developed. ⑶CQULAF4/LAR4 primers and TaqMan probe were designed based on the sequence of specific target fragment of HLB pathogen. Then HLB were detected by the Real time Quantitative PCR technique. It is more sensitive, simpler, faster but more expensive than the conventional PCR. ⑷Candidatus Liberibacter asiaticus is uncultured G-bacterial species. The whole DNA of both the bacteria and the citrus plant is not ideal positive control for its short storage time. Recombined plasmid of HLB target fragment not only was used as ideal positive control with safty but also can be used as a norm for quantitative detection of Ca. L. asiaticus. ⑸The adding of macromolecular stabilizer (MS) can promote the stabilization of pre-mixed PCR reagents. By vaccum freeze-drying treatment, it can be preserved at least six months at room temperature. Conclusion: Detection system of Candidatus Liberibacter asiaticus with rapidness, accuracy, and stabilization was established. Detection Kit based on detection system as a practical tool can apply to disease diagnosis for establishment of disease-free area of citrus production, identification of citrus diseases with suspicious lesions, and precautionary monitoring of HLB dynamically.
研究目的:本文对柑桔黄龙病菌核酸分子检测技术和生物大分子常温贮藏技术进行了系统的研究,旨在建立起快速准确稳定安全的柑桔黄龙病菌分子检测技术体系,实现柑桔样品的快速检验,为柑桔非疫区生产建设和病害预警监测提供一种分子检测技术和实用工具。
研究方法:利用CTAB 法,试剂盒法和浸泡过滤法提取柑桔黄龙病菌的核酸DNA;利用引物和探针设计软件Primer Premier 5.0 进行引物和探针的设计;PCR同步扩增柑桔叶面腐生菌,柑桔溃疡病菌和衰退病菌等等植物细菌和病原物来验证检测的特异性;PCR 扩增含有靶片段的质粒DNA 测定检测的灵敏度;通过在不同DNA 扩增仪和温度控制方式下的扩增程序优化测定检测的稳定性。将靶片段与克隆载体pMD-18T 连接后,用CaCl2法转化大肠杆菌(E. coli,JM109),然后通过测序来验证PCR 扩增产物的准确性;以重组质粒和转化的大肠杆菌构建了无害化阳性参照体系。采用预混PCR 反应液中加入生物活性分子稳定剂并冷冻干燥固相化处理的方法研制出能常温贮存的方便有效的检测试剂盒。利用优化的分子检测体系对江西、浙江、广西、湖南、重庆和四川等地采集的柑桔样品进行了PCR实际检测。利用iCycler iQ定量PCR 仪对柑桔黄龙病的定量检测做了初步的研究。
研究结果:
1) 通过比较研究不同制样方法对检测效果的影响,确定浸泡过滤法能有效地排除植物色素、蛋白质、多糖等物质对PCR 反应的干扰,制备适合于PCR 反应的靶细菌DNA,避免PCR 反应的假阴性发生。
2) 根据柑桔黄龙病菌核糖体蛋白基因设计筛选了不同的引物对,筛选出特异性强稳定性好灵敏度高的引物对CQULAF3/CQULAR3,并由此建立了准确、快速、灵敏、稳定、安全的柑桔黄龙病菌PCR 检测体系。
3) 在引物CQULAF3/CQULAR3 扩增产物中设计了引物CQULAF4/CQULAR4,
Citrus Huanglongbing Disease (HLB), caused by an uncultured phloem-restricted, G-bacterium, is one of severe quarantine diseases domestically and abroad. After the citrus trees are infected, the yield and quality are affected, even the whole is dead. At present the appropriate method is to eradicate and burn infected plant material. In order to prevent the disease from spreading in citrus plant area of China we should develop disease-free area of citrus production. Therefor it is important to do research on detection technique. There are some disadvantages i.e. low specificity and sensitivity in the routine detection techniques. It is hard to meet the Quaratine requirement and conduct presentation diagnosis of HLB. The detection according to nucleic acid molecule is rapid, sensitive and special and can meet modern plant quarantine requirement domestically and abroad.
Objective: This thesis aims at developing detection system of DNA molecule with specificity, rapidness, accuracy and stabilization and providing practical tool of disease diagnosis for establishment of disease-free area and precautionary monitoring of HLB through the research on the molecule detection techniques of HLB and diagnosis kit stored at room temperature systemically.
Methods: First of all, DNA of Candidatus Liberibacter asiaticus was extracted by the methods of CTAB,reagent kits and soaking-filtration. Primer pairs and a probe were designed using software Primer Premier 5.0. The specificity was validated through PCR detection of the saprophytic microorganism of citrus leaf surface, Tristeza virus and bacteria of Xac etc plant pathogens or G-/ G+ bacteria. The sensitivity of PCR was determinated by amplification of the bacteria DNA and target sequence inserted in recombinant plasmid solution with ten-fold serial dilution. PCR profiles were adjusted by using different type of Thermocycler in different temperature control model, which ensured the stability of PCR detection. After being inserted into clone vector and recombinant plasmid transformed into the E. coli strain (JM109), the target DNA fragment was sequenced, and amplification fidelity of PCR was verified by sequences alignment using BLASTn. Then a harmless positve reference system was constructed with recombinant plasmid DNA. Solidifying PCR reagent kit was produced by adding macromolecular stabilizer (MS) into PCR mixed reagents and vacuum freeze-drying treatment. Field samples with or without disease symptom of citrus collected from
Guangxi, Zhejiang, Jiangxi, Sichuan provinces and Chongqing area were detected by PCR assay. The primary study on quantitative detection of HLB pathogen (Ca.L.asiaticus) was made by the iCycleriQ Realtime detection system. Experiment Results: ⑴DNA of Ca.L.asiaticus extraction methods were compared. Among them the soaking-filtration is the best one, which remove plant pigment, protein and polysaccharide totally, pure target DNA was obtained and false negativity of PCR reaction was avoided effectively. ⑵Specific primers (CQULAF3/LAR3) which have good specificity, sensitivity and stability for HLB detection, were designed based on published sequence encoding a ribosome protein gene and detection system of DNA molecule with rapidness, accuracy, stabilization have been developed. ⑶CQULAF4/LAR4 primers and TaqMan probe were designed based on the sequence of specific target fragment of HLB pathogen. Then HLB were detected by the Real time Quantitative PCR technique. It is more sensitive, simpler, faster but more expensive than the conventional PCR. ⑷Candidatus Liberibacter asiaticus is uncultured G-bacterial species. The whole DNA of both the bacteria and the citrus plant is not ideal positive control for its short storage time. Recombined plasmid of HLB target fragment not only was used as ideal positive control with safty but also can be used as a norm for quantitative detection of Ca. L. asiaticus. ⑸The adding of macromolecular stabilizer (MS) can promote the stabilization of pre-mixed PCR reagents. By vaccum freeze-drying treatment, it can be preserved at least six months at room temperature. Conclusion: Detection system of Candidatus Liberibacter asiaticus with rapidness, accuracy, and stabilization was established. Detection Kit based on detection system as a practical tool can apply to disease diagnosis for establishment of disease-free area of citrus production, identification of citrus diseases with suspicious lesions, and precautionary monitoring of HLB dynamically.
引文
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