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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2015, Vol. 41 Issue (1): 25-33    DOI: 10.3785/j.issn.1008-9209.2014.06.261
生物科学与技术     
基于序列特异扩增区域标记的栗疫菌巢式PCR检测技术
麻文建, 郑磊, 张静, 刘洋, 朱天辉*
四川农业大学林学院,四川 雅安625014
Development of nested-PCR for detection of Cryphonectria parasitica based on the marker of sequence characterized amplified region
Ma Wenjian, Zheng Lei, Zhang Jing, Liu Yang, Zhu Tianhui*
(College of Forestry, Sichuan Agricultural University, Ya'an 625014, Sichuan, China)
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摘要： 为建立栗疫菌(Cryphonectria parasitica)快速、准确的检测技术,利用随机扩增多态性DNA技术(random amplified polymorphic DNA, RAPD)对不同来源地的栗疫菌和其他真菌分离物进行PCR扩增,筛选出栗疫菌的RAPD特异片段SCQ494。将特异RAPD片段SCQ494进行分离、回收,与载体pMD19-T载体连接,转化至大肠埃希菌进行培养,对目标片段克隆测序。根据测序结果,用Primer 5.0软件设计了序列特异扩增区域(sequence characterized amplified region, SCAR)引物CQ1/CQ2和巢式PCR引物CR1/CR2。利用引物CQ1/CQ2通过常规PCR对不同来源地供试栗疫菌可扩增出1条约1 420 bp的条带,对其他供试菌株DNA的PCR产物均无此条带,检测灵敏度为3 pg/μL的基因组DNA; 而以引物CQ1/CQ2为外圈引物和引物CR1/CR2为内圈引物进行的巢式PCR可从栗疫菌基因组DNA中扩增出1条大小约875 bp 的条带,灵敏度达到30 fg/μL,是常规PCR的1000倍。验证实验表明巢式PCR 可以从发病程度不同的栗树枝干和在自然感染的栗树枝条中检测出栗疫菌。
Abstract: Chestnut blight, caused by Cryphonectria parasitica, is a destructive disease on chestnut trees as well as an important international disease in the world. At the end of the 19th century and the beginning of the 20th century, this pathogen dispersed rapidly and nearly killed all the American chestnut trees. Cryphonectria parasitica belongs to ascomycetes, which mainly caused considerable damage to species of genus Castanea, such as C. sativa, C. henryi, C. dentata, and so on. In recent years, the chestnut blight tends to be aggravated and has caused tremendous loss in the ecology and economy. The traditional detection methods for C. parasitica are time-consuming, tedious, laborious, low sensitivity and accuracy. However, previous studies also did not have a lot of detection reports about C. parasitica. Therefore, rapid and efficient detection of C. parasitica is essential for undertaking appropriate and timely disease management measures. In the present study, polymerase chain reaction (PCR) assay had been developed for accurate and sensitive detection of some plant pathogens, which are much faster and more specific than traditional detection methods. The objective of this study was to develop a sequence characterized amplified region (SCAR) marker and PCR detection of C. parasitica. Randomly amplified polymorphic DNA (RAPD) was used to detect DNA polymorphisms between C. parasitica and other strains, and the specific RAPD fragment of C. parasitica was purified and inserted into pMD19-T vector that was transformed into Escherichia coli and cloned and sequenced. Based on the sequence of the RAPD marker, SCAR primers and the nested-PCR primers were designed and synthesized. Then the specificity and sensitivity of primers were verified. The results showed that primer S494 generated a polymorphic pattern displaying a 1 400 bp DNA fragment (SCQ494) specific for C. parasitica, but not from any other strains tested. Based on the sequence of SCQ494, the specific SCAR primers CQ1/CQ2 and the nested-PCR primers CR1/CR2 were designed by the aid of the software Primer Premier 5.0. The regular PCR product of all strains of C. parasitica showed a unique fragment of 1 420 bp with primers CQ1/CQ2; the nested-PCR primers CR1/CR2 amplified only a unique 875 bp band from all C. parasitica and there was no amplification from other strains tested. The detection sensitivity with primer set CQ1/CQ2 was 3 pg/μL for genomic DNA of C. parasitica in 25 μL reaction solution. In contrast, nested-PCR with CQ1/CQ2 as the first round primers and CR1/CR2 as the second round primers increased sensitivity to 30 fg/μL for genomic DNA of C. parasitica. In addition, C. parasitica could be specifically detected by nested-PCR assay from diseased plant tissues collected from field and artificial inoculation branches. In conclusion, nested-PCR for detection of C. parasitica has been developed based on the use of a SCAR marker and has a great significance for the prevention and control of chestnut blight. This is the first report on generation of SCAR markers for detection of C. parasitica and the result of the study may provide an important reference for the detection of other phytopathogen.
出版日期: 2015-01-20
CLC:  Q 939.95  
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麻文建
郑磊
张静
刘洋
朱天辉

引用本文:

麻文建, 郑磊, 张静, 刘洋, 朱天辉. 基于序列特异扩增区域标记的栗疫菌巢式PCR检测技术[J]. 浙江大学学报(农业与生命科学版), 2015, 41(1): 25-33.

Ma Wenjian, Zheng Lei, Zhang Jing, Liu Yang, Zhu Tianhui. Development of nested-PCR for detection of Cryphonectria parasitica based on the marker of sequence characterized amplified region. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(1): 25-33.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2014.06.261    或    http://www.zjujournals.com/agr/CN/Y2015/V41/I1/25

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