Establishment and Preliminary Application of a Quantitative Realtime PCR Method for Detection of Conidia of Ustilaginoidea virens

doi:10.3969/j.issn.10017216.2012.04.017

Chinese Journal of Rice Science ›› 2012, Vol. 26 ›› Issue (4): 500-505.DOI: 10.3969/j.issn.10017216.2012.04.017

• Experimental Techniques • Previous Articles Next Articles

Establishment and Preliminary Application of a Quantitative Realtime PCR Method for Detection of Conidia of Ustilaginoidea virens

ZHENG Jing ^1，2， ZHANG Zhen ^2，*， JIANG Hua ²， WANG Yanli ²， CHAI Rongyao ²， QIU Haiping ²， MAO Xueqin ²，WANG Jiaoyu²， DU Xinfa ²， LAN Zhaohui ³， SUN Guochang ^2，*

¹College of Chemistry and Life Science， Zhejiang Normal University， Jinhua 321004， China ; ²State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control， Institute of Plant Protection Microbiology，Zhejiang Academy of Agricultural Sciences， Hangzhou 310021， China; ³Xiangshan Plant Protection and Quarantine Station, Xiangshan 315700, China;

Received:2011-09-16 Revised:2012-03-20 Online:2012-07-10 Published:2012-07-10
Contact: ZHANG Zhen2，*，SUN Guochang2，*

稻曲病菌分生孢子实时PCR定量检测方法的建立及初步应用

郑静^1，2 ,张震^2，* ,姜华² ,王艳丽² ,柴荣耀² ,邱海萍² ,毛雪琴² ,王教瑜² ,杜新法²,赖朝晖³,孙国昌^2，*

¹浙江师范大学化学与生命科学学院，浙江金华321004； ²浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地/浙江省农业科学院植物保护与微生物研究所，浙江杭州310021； ³浙江省象山县植物保护检疫站，浙江象山 315700；

通讯作者: 张震2，* ,孙国昌2，*
基金资助:
浙江省农业厅三农五方项目（2009）；宁波市粮食生产功能区项目（2009C10038）；浙江省公益技术研究农业项目（2011C22008）；浙江省优先主题资助项目（2011C12022）。

Abstract

Abstract: A realtime PCR quantitative detection technology was established by using two pairs of specific primers designed according to the internal transcribed spacer (ITS) sequences of Ustilaginoidea virensand their corresponding TaqMan probes. The results showed that the combination of the specific primers and probe of uvr292rf/uvr397rr/uvrp333 was highly sensitive and specific in the quantification of U. virens with a minimum detection limit of 24 fg. A linear model between Ct and common logarithm of spores number was established by realtime PCR with the templates of DNA from diluted spores solution and the theoretical detected limit was 0.67 spore. According to the quantitative detection of three air samples collected in paddy field at various time with this linear model, the quantity of this fungus conidia fluctuated monthly.

Key words: Ustilaginoidea virens, conidia, realtime PCR, internal transcribed spacer (ITS)

摘要： 根据稻曲病菌核糖体转录间隔区（ITS）序列的特异性，设计了2对引物及相应TaqMan探针用于建立稻曲病菌实时PCR定量检测技术。结果表明，uvr292rf/uvr397rr/uvrp333组合建立的体系可特异性检测出稻曲病菌，扩增基线平整，指数扩增期明显，重现性好，最低可检出24 fg的稻曲病菌基因组DNA模板量；以梯度稀释分生孢子液提取的基因组DNA为模板，建立孢子数量常用对数值与Ct值的线性关系，理论上最低可检测出0.67个分生孢子。对3个时间点的田间孢子捕捉样品进行检测，结果显示不同月份间稻曲病菌孢子数量存在差异。

关键词: 稻曲病菌, 分生孢子, 实时PCR, 核糖体转录间隔区

CLC Number:

ZHENG Jing1，2， ZHANG Zhen2，*， JIANG Hua2， WANG Yanli2， CHAI Rongyao2， QIU Haiping2， MAO Xueqin2，WANG Jiaoyu2， DU Xinfa2， LAN Zhaohui3， SUN Guochang2，*. Establishment and Preliminary Application of a Quantitative Realtime PCR Method for Detection of Conidia of Ustilaginoidea virens [J]. Chinese Journal of Rice Science, 2012, 26(4): 500-505.

郑静1，2 ,张震2，* ,姜华2 ,王艳丽2 ,柴荣耀2 ,邱海萍2 ,毛雪琴2 ,王教瑜2 ,杜新法2 ,赖朝晖3 ,孙国昌2，*. 稻曲病菌分生孢子实时PCR定量检测方法的建立及初步应用[J]. 中国水稻科学, 2012, 26(4): 500-505.

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Establishment and Preliminary Application of a Quantitative Realtime PCR Method for Detection of Conidia of Ustilaginoidea virens

稻曲病菌分生孢子实时PCR定量检测方法的建立及初步应用

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