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。
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
.
DOI: 10.3969/j.issn.10017216.2012.04.017
A realtime 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 realtime 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.
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