基于锁式探针的番茄溃疡病菌实时荧光PCR快速检测

doi:10.3864/j.issn.0578-1752.2014.05.007

摘要/Abstract

摘要： 【目的】从口岸进境番茄种子中检测番茄溃疡病菌（Clavibacter michiganensis subsp. michiganensis，CMM），一直以来都受检测周期的限制，快速、特异地从种子中检测该病原细菌需要新的方法。研究在锁式探针扩增方式上，选择连接酶依赖的PCR扩增方式，并结合实时荧光PCR技术，旨在建立番茄溃疡病菌基于锁式探针技术的实时荧光PCR快速检测方法，为口岸番茄种子快速检疫提供技术支持。【方法】根据番茄溃疡病菌的一段特异基因Pat-1序列，设计锁式探针的T1和T2臂，使之与CMM的特异性片段碱基序列互补，再按照锁式探针的设计原则设计锁式探针和扩增引物以及荧光探针。试验时，先将锁式探针与CMM以及参照菌株的DNA模板在DNA连接酶作用下分别进行环化连接，再用核酸外切酶Ⅰ和核酸外切酶Ⅲ消化未环化的线性锁式探针，最后以环化的锁式探针为模板，在扩增引物的作用下进行实时荧光PCR试验。建立CMM基于锁式探针技术的实时荧光PCR检测方法，分别比较该方法的特异性和灵敏度与常规PCR方法的差异，并用该方法对收集的来自日本、韩国和中国台湾的番茄种子以及国内采集的共45份样品进行检测。【结果】基于锁式探针技术的实时荧光PCR检测方法能够从供试的菌株中特异性地检出CMM，在供试的10种菌株中，只有靶标细菌能被特异性地检测到阳性，空白对照和其他参试菌株均没有荧光信号的增加，检测为阴性。比较该检测方法与常规PCR方法，其特异性和常规PCR方法一致。该检测方法检测灵敏度高，DNA最低浓度检测为50 fg•μL-1，而常规PCR方法检测DNA最低浓度为5 pg•μL-1，灵敏度高于常规PCR两个数量级。对收集的样品进行检测，结果显示，45份样品中共有5份样品CMM检测结果为阳性，分别是日本的番茄种子样品2份（编号Jap1214、Jap1102），永泰采集的样品2份（编号为Yongt1001、Yongt1002）和闽清采集的样品1份（编号为Minq1001）。【结论】建立的基于锁式探针技术的荧光PCR方法具有高度的特异性和灵敏度，应用该检测方法对收集的进境番茄种子进行检测，可以直接从番茄种子中检测到CMM，该方法适合口岸番茄种子CMM的快速检测，有较好的口岸检疫实际应用价值。

关键词: 番茄细菌性溃疡病菌 , 锁式探针 , 实时荧光PCR , 超分支滚环扩增 , 快速检测 , 番茄种子

Abstract: 【Objective】Detection of tomato canker (Clavibacter michiganensis subsp. michiganensis, CMM) from imported tomato seeds has been restricted to the detection time, therefore, a new approach is needed to detect CMM rapidly and specifically from tomato seeds. In this study, combined with real-time PCR technology, the ligase-dependent PCR amplification was carried out in amplification way of padlock probe. The objective of this study is to establish the method of real-time PCR based on the padlock probe for detecting CMM, and to provide technical supports for entry-exit phytosanitary. 【Method】 T1 and T2 arms of the padlock probe, which were complementary to the specific nucleotide sequence of CMM, were designed based on the sequence of the unique hypothetic protein gene Pat-1 in complete genome of CMM. The universal primers and TaqMAN fluorescent probes were also designed according to the design principles of padlock probes. During the test, firstly, padlock probe was connected with the DNA of CMM and other reference strains under the action of the DNA enzyme, respectively, then, the connected product padlock probe was digested with exonucleaseⅠand Ⅲ to remove uncyclized linear padlock probe, finally, real-time PCR amplification was tested with the Cyclized padlock probes as a DNA template. The detection system for CMM by using real-time PCR based on padlock probe was established, the specificity and sensitivity of this method were compared with the conventional PCR method, respectively, and a total of 45 tomato samples were detected using this method, which were collected from Japan, Korea and China’s Taiwan, as well as the main land of China. 【Result】 CMM could be detected specifically from the 10 tested strains with real-time PCR based on padlock probe method, only the target bacteria could be specifically detected positive, the blank control and other strains were detected negative. The specificity was consistent with the conventional PCR method. The detection sensitivity of real-time PCR based on padlock probe for DNA concentration was about 50 fg•μL-1, while the sensitivity of conventional PCR method was about 5 pg•μL-1, the former was 100 times higher than the latter. To detect tomato samples for CMM, 5 samples were tested positive from 45 samples, among which 2 samples were from Japan (Jap1214, Jap1102), 2 samples from Yongtai (Yongt1001, Yongt1002) and 1 sample from Minqing (Minq1001). 【Conclusion】The assay of real-time PCR based on padlock probe was specific and sensitive, and the detection method was used to test the entry tomato seeds collected, CMM could be detected directly from tomato seeds. This method would be suitable for CMM rapid detection of tomato seeds imported, and would be an effective tool for detecting CMM in entry-exit phytosanitary.

Key words: Clavibacter michiganensis subsp. michiganensis , padlock probe , real-time PCR , hyperbranched rolling circle amplification , rapid detection , tomato seed

王念武1, 2, 王婷1, 沈建国2, 胡方平1. 基于锁式探针的番茄溃疡病菌实时荧光PCR快速检测[J]. 中国农业科学, 2014, 47(5): 903-911.

WANG Nian-Wu-1, 2 , WANG Ting-1, SHEN Jian-Guo-2, HU Fang-Ping-1. Rapid Detection for Clavibacter michiganensis subsp. michiganensis Using Real-Time PCR Based on Padlock Probe[J]. Scientia Agricultura Sinica, 2014, 47(5): 903-911.

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参考文献

[1]Chang R J, Ries S M, Pataky J K. Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants. Phytopathololgy, 1991, 81: 1276-1281.

[2]Gitaitis R D, Beaver R W, Voloudakis A E. Detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato transplants. Plant Disease, 1991, 75(8): 834-838.

[3]Gleason M L, Gitaitis R D, Ricker M D. Recent progress in understanding and controlling bacterial canker of tomato in Eastern North America. Plant Disease, 1993, 77(11): 1069-1076.

[4]Jahr H, Bahro R, Bruger A, Ahlemeyer J, Eichenlaub R. Interactions between Clavibacter michiganensis and its host plants. Environmental Microbiology, 1999, 1(2): 113-118.

[5]Luo L X, Walters C, Bolkan H, Liu X L, Li J Q. Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay. Plant Pathology, 2008, 57: 332-337.

[6]Fatmi M, Schaad N W. Survival of Clavibacter michiganensis subsp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco. Plant Pathology, 2002, 51: 149-154.

[7]Francis D M, Kabelka E, Bell J, Franchino B, St. Clair D. Resistance to bacterial canker in tomato (Lycopersicon hirsutum LA407) and its progeny derived from crosses to L.esculentum. Plant Disease, 2001, 85: 1171-1176.

[8]Smith I M, McNamara D G, Scot M, Holderness P R. Quarantine Pest for Europe[DB/CD]. Wallingford, UK: EPPO/CABI, 1997.

[9]De León L, Siverio F, Rodriguez A. Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation. Journal of Microbiological Methods, 2006, 67: 141-149.

[10]Kaneshiro W S, Mizumoto C Y, Alvarez A M. Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophytes using ELISA, Biolog and 16S rRNA sequencing. European Journal of Plant Pathology, 2006, 116(1): 45-56.

[11]Veena M S, van Vuurde J W L. Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato. Journal of Microbiological Methods, 2002, 49: 11-17.

[12]Bach H J, Jessen I, Schloter M, Munch J C. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies. Journal of Microbiological Methods, 2003, 52: 85-91.

[13]Nogva H K, Dromtorp S M, Nissen H, Rudi K. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. BioTechniques, 2003, 34(4): 804-813.

[14]Dreier J, Bermpohl A, Eichenlaub R. Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopatohology, 1995, 85: 462-468.

[15]夏明星, 赵文军, 马青, 朱水芳. 番茄细菌性溃疡病菌的实时荧光PCR检测. 植物病理学报, 2006, 36(2): 152-157.

Xia M Z, Zhao W J, Ma Q, Zhu S F. Rapid detection of tomato bacterial canker (Clavibacter michiganensis subsp. michiganensis) with real-time fluorescent PCR. Acta Phytopathologica Sinica, 2006, 36(2): 152-157. (in Chinese)

[16]Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary B P, Landegren U. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science, 1994, 265(5181): 2085-2088.

[17]Thomas D C, Nardone G A, Randall S K. Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction. Archives of Pathology & Laboratory Medicine, 1999, 123(12): 1170-1176.

[18]Zhang D Y, Brandwein M, Hsuih T C, Li H B. Amplification of target-specific ligation-dependent circular probe. Gene, 1998, 211(2): 277-285.

[19]刘倩, 李蓓, 文景芝, 陈洪俊, 葛建军. 基于锁式探针的滚环扩增技术在植物病原检测中的应用. 植物检疫, 2009, 23(3): 38-41.

Liu Q, Li B, Wen J Z, Chen H J, Ge J J. Application of rolling circle amplification based on padlock probe in plant pathogen detection. Plant Quarantine, 2009, 23(3): 38-41. (in Chinese)

[20]赵春燕, 易世红, 李凡. 网状分枝扩增技术快速检测大肠埃希菌0157: H7及其他产志贺样毒素大肠埃希菌. 吉林大学学报: 医学版, 2006, 32(1): 18-22.

Zhao C Y, Yi S H, Li F. Rapid detection of E. coli O157: H7 and other Shiga toxin-producing E. coli using ramification amplification method. Journal of Jilin University: Medicine Edition, 2006, 32(1): 18-22. (in Chinese)

[21]Li J, Deng T, Chu X, Yang R, Jiang J, Shen G, Yu R. Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Analytical Chemistry, 2010, 82(7): 2811-2816.

[22]Su Q, Xing D, Zhou X. Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation. Biosensors and Bioelectronics, 2010, 25: 1615-1621.

[23]Yan J, Song S, Li B, Zhang Q, Huang Q, Zhang H, Fan C. An on-nanoparticle rolling-circle amplification platform for uItrasensitive protein detection in biological fluids. Small, 2010, 6(22): 2520-2525.

[24]Cheng W, Yan F, Ding L, Ju H, Yin Y. Cascade signal amplication strategy for subattomolar protein detection by rolling circle amplification and quantum dots tagging. Analytical Chemistry, 2010, 82(8): 3337-3342.

[25]郭艳玲, 刘洋, 姜广路, 时广利, 宋长兴, 傅瑜. 超分支滚环扩增技术快速检测结核分枝杆菌感染的临床应用价值. 结核病与胸部肿瘤, 2012(2): 89-91.

Guo Y L, Liu Y, Jiang G L, Shi G L, Song C X, Fu Y. Clinical application on rapid detection of Mycobacterium tuberculosis using hyperbranched rolling circle amplification. Tuber & Thor Tumor, 2012(2): 89-91. (in Chinese)

[26]Szemes M, Bonants P, de Weerdt M, Baner J, Landegren U, Schoen C D. Diagnostic application of padlock probes-multiplex detection of plant pathogens using universal microarrays. Nucleic Acids Research, 2005, 33(8): e70.

[27]黄冠军, 殷幼平, 张仑, 李小焦, 葛建军, 陈洪俊, 王中康. 柑橘溃疡病菌滚环扩增检测体系的建立. 微生物学报, 2008, 48(3): 375-379.

Huang G J, Yin Y P, Zhang L, Li X J, Ge J J, Chen H J, Wang Z K. Detection system for Xanthomonas axonopodis pv. citri using rolling circle amplification. Acta Microbiologica Sinica, 2008, 48(3): 375-379. (in Chinese)

[28]Lizardi P M, Huang X H, Zhu Z R, Ward P B, Thomas D C, Ward D C. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genetics, 1998, 19: 225-232.

[29]Wang B, Potter S J, Lin Y, Cunningham A L, Dwyer D E, Su Y, Ma X, Hou Y, Saksena N K. Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification. Journal of Clinical Microbiology, 2005, 43(5): 2339-2344.