应用锁式探针结合斑点杂交技术检测甜瓜细菌性叶斑病

Development of Padlock Probe Combined with Dot-Blot Hybridization Based Methods for Detection of Bacterial Spot of Melon Leaves

【目的】甜瓜细菌性叶斑病是危害甜瓜的重要种传细菌病害,是由丁香假单胞杆菌流泪病致病变种甜瓜菌株(Pseudomonas syringae pv.lachrymans)引起的。论文旨在建立高效、快捷、操作简单的检测技术以防止此病原菌传播。【方法】以Gen Bank公布的甜瓜细菌性叶斑病菌的甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因序列为靶标,设计甜瓜细菌性叶斑病菌特异性锁式探针,建立锁式探针与斑点杂交技术结合的检测体系。以保存的目的菌株为材料,提取DNA作为模板进行锁式探针连接反应、酶切反应和扩增反应,并将锁式探针连接反应、酶切反应和扩增反应的反应温度和反应时间分别进行优化。利用优化的锁式探针连接反应、酶切反应和扩增反应分别对健康的甜瓜种子、带有甜瓜细菌性叶斑病的甜瓜种子、无菌水及25株其他参试菌株进行检测,测定该体系的特异性。将甜瓜细菌性叶斑病菌的DNA按10倍梯度稀释,依次稀释为1 ng·μL^(-1)、100 pg·μL^(-1)、10 pg·μL^(-1)、1 pg·μL^(-1)、100 fg·μL^(-1)、10 fg·μL^(-1)和1 fg·μL^(-1)作为模板,利用优化的锁式探针连接反应、酶切反应和扩增反应,测定灵敏度。将探针与斑点杂交技术结合建立高通量检测体系,将上述反应过程中的扩增产物固定于尼龙膜上,将锁式探针上Zipcode序列的反向互补序列合成c Zipcode(检测探针),检测探针(c Zipcode)用地高辛标记后与产物进行杂交。锁式探针结合斑点杂交技术分别进行特异性检测和灵敏度测定。探针与斑点杂交技术结合建立的高通量检测体系进行人工模拟种子带菌检测,进一步验证该体系的可靠性。利用建立的高通量检测方法对205份市售疑似带病的甜瓜种子进行检测。【结果】锁式探针特异性测定结果表明,26株甜瓜细菌性叶斑病菌均能得到一条105 bp的特异性条带,而剩余25株参试菌株及无菌水均无扩增产物产生。灵敏度测定结果表明,当目的菌株的浓度稀释为1 pg·μL^(-1)时均能检测到一条105 bp的特异性条带,所以探针的检测灵敏度为1 pg·μL^(-1)。探针与斑点杂交技术结合建立的检测甜瓜细菌性叶斑病菌高通量体系能将甜瓜细菌性叶斑病与所有的参试菌种区分开,26株甜瓜细菌性叶斑病菌杂交后出现了显色反应,而25株参试菌株及无菌水均没有发生显色。锁式探针结合斑点杂交的灵敏度检测同样达到1 pg·μL^(-1)。将锁式探针结合斑点杂交进行人工模拟种子带菌检测,能将1粒带菌种子从1 000粒健康的种子检测出来,模拟种子带菌检测率都能达到0.1%(1/1 000)。从205份市售甜瓜种子中成功检测到7份市售种子带菌。将带菌种子分别加入一定量的无菌水浸泡4 h,提取悬液DNA,将悬液DNA进行PCR扩增后测序,NCBI比对后确定为甜瓜细菌性叶斑病菌。【结论】基于锁式探针结合斑点杂交技术的检测体系能够快速、准确地识别甜瓜细菌性叶斑病。

【Objective】 Bacterial spot of melon leaves caused by Pseudomonas syringae pv. lachrymans is distributed widely in the world and inflicts different degrees of damage. P. syringae pv. lachrymans is a typical seed-borne pathogen. The objective of this study is to build effective, commercially viable and convenient detecting technologies and to prevent the spread of this pathogen.【Method】The house-keeping gene of DNA glyceraldehyde-3-phosphate dehydrogenase（GAPDH） was selected as the target gene, the specific Padlock probe was designed for bacterial leaf spot of melon, and a bacterial detection system based on Padlock probe combined with dot-blot hybridization was developed. The target strain was selected as experimental materials, and DNA was extracted as a template for ligation reaction, enzyme-cleavage reaction and amplification reaction. The reaction temperature and reaction time of the ligation reaction, enzyme-cleavage reaction and amplification reaction were optimized. The optimized reactions were tested for healthy melon seeds, melon seeds with bacterial spot of melon leaves, sterile water and 25 experimental strains. In order to determine the sensitivity of Padlock probe, DNA of the target strain was diluted to 1 ng·μL^（-1）, 100 pg·μL^（-1）, 10 pg·μL^（-1）, 1 pg·μL^（-1）, 100 fg·μL^（-1）, 10 fg·μL^（-1） and 1 fg·μL^（-1） as the templates. Sensitivity was determined by optimized ligation reaction, enzyme-cleavage reaction and amplification reaction. Padlock probe combined with dot-blot hybridization was developed, the amplification product in the course of the reaction was fixed on the nylon membrane, the reverse complementary sequence of the Zipcode sequence was synthesized into c Zipcode（detection probe）, the detection probe（c Zipcode） was labeled with digoxigenin and hybridized with the amplified product. Padlock probe combined with dot-blot hybridization techniques were used for specific detection and sensitivity. Artificial infestation seeds were detected to further verify the reliability of the system. A total of 205 commercially melon seeds with suspected disease were detected by high throughput detection method.【Result】The specificity of the Padlock probe showed that a specific band of 105 bp was got from 26 strains of bacterial spot of melon leaves, while the remaining 25 strains and sterile water were not amplified products. The results of sensitivity showed that the target strain was diluted to 1 pg·μL^（-1） and got a specific band of 105 bp, so the detection sensitivity of the Padlock probe was 1 pg·μL^（-1）. Padlock probe could distinguish bacterial spot of melon leaves from all other experimental strains and its sensitivity could be up to 1 pg·μL^（-1）.Twenty-six strains of bacterial spot of melon leaves had color reaction, the remaining 25 strains and sterile water did not have color reaction. The sensitivity of Padlock probe combined with dot-blot hybridization also could be up to 1 pg·μL^（-1）. Padlock probe combined with dot-blot hybridization could detect one bacterial seed from 1 000 healthy seeds, and the detection rate reached 0.1%（1/1 000）. Seven commercially seed-borne bacteria were successfully detected from 205 commercially melon seeds. The seven seed-borne bacteria, respectively, were soaked in sterile water for 4 h, DNA was extracted for PCR amplification and sequencing, the results of DNA sequencing were compared with NCBI and verified the bacterial spot of melon leaves. 【Conclusion】Detecting system based on Padlock probe combined with dot-blot hybridization could detect P. syringae pv. lachrymans from sweet melon fast and accurately.