| 烟草拟茎点霉快速检测方法的建立 |
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| 引用本文： | 邢国珍,孙帅,李旭,邱睿,程琨,李博,陈佳敏,何雷,李淑君,郑文明.烟草拟茎点霉快速检测方法的建立[J].烟草科技,2022,55(12):19-24. |
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| 作者姓名： | 邢国珍 孙帅 李旭 邱睿 程琨 李博 陈佳敏 何雷 李淑君 郑文明 |
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| 作者单位： | 1.河南农业大学生命科学学院，郑州市金水区农业路63号 4500022.河南省农业科学院烟草研究所 黄淮烟区烟草病虫害绿色防控重点实验室，河南省许昌市魏都区永昌大道与青梅路交叉口 4610003.中国烟草总公司河南省公司，郑州市金水区商务外环15号 450018 |
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| 基金项目： | 河南省高等学校重点科研项目“烟草镰刀菌根腐病发生相关根系微生物群落解析与生防菌筛选研究”20B210009中国烟草总公司河南省公司科技项目“烟草根腐病害发生的微生物群落分析和生态风险预警研究”2020410000270012河南农业大学教学实验室开放创新项目“烟草真菌类病害的快速分子检测方法研究”KF201914国家自然科学基金青年基金项目“硒防治小麦茎基腐病的生理效应及机制研究”32102230 |
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| 摘 要： | 为实现烟草拟茎点霉的快速准确检测，利用真菌rDNA-ITS通用引物对拟茎点霉和其他10种烟草病原菌基因组DNA进行了PCR扩增、片段回收及DNA测序，在序列比对基础上设计了两对特异性检测引物，并鉴定了引物的检测灵敏度和特异性，同时优化建立了PCR反应体系。结果表明，在退火温度为60 ℃时，两对特异性引物P2F/P3R和P3F/P3R从烟草拟茎点霉DNA样本中分别扩增出来420 bp和381 bp的特异性条带，而在其他10种烟草病原菌DNA以及阴性对照中均不能扩增到任何条带。两对特异性引物的检测灵敏度均达到10 pg/μL。建立的基于两对特异性引物P2F/P3R和P3F/P3R的分子检测方法可实现对病原菌烟草拟茎点霉的快速准确检测。
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| 关 键 词： | 烟草 拟茎点霉 特异性引物 分子检测 |
| 收稿时间： | 2022-06-06 |
A rapid detection method for Phomopsis sp. in tobacco |
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| XING Guozhen,SUN Shuai,LI Xu,QIU Rui,CHENG Kun,LI Bo,CHEN Jiamin,HE Lei,LI Shujun,ZHENG Wenming.A rapid detection method for Phomopsis sp. in tobacco[J].Tobacco Science & Technology,2022,55(12):19-24. |
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| Authors: | XING Guozhen SUN Shuai LI Xu QIU Rui CHENG Kun LI Bo CHEN Jiamin HE Lei LI Shujun ZHENG Wenming |
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| Affiliation: | 1.College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, China2.Key Laboratory for Green Preservation & Control of Tobacco Diseases and Pests in Huanghuai Growing Area, Tobacco Research Institute, Henan Academy of Agricultural Sciences, Xuchang 461000, Henan, China3.Henan Provincial Tobacco Company of CNTC, Zhengzhou 450018, China |
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| Abstract: | To detect Phomopsis sp. in tobacco rapidly and accurately, PCR amplification, fragment recovery and DNA sequencing were carried out on the genomic DNA of Phomopsis sp. and 10 other tobacco pathogens by using fungal rDNA-ITS universal primers. Two pairs of specific detection primers were designed, and the detection sensitivity and specificity of the primers were evaluated. Furthermore, the PCR reaction system was optimized. The results showed that at the annealing temperature of 60 ℃, two pairs of specific primers P2F/P3R and P3F/P3R amplified specific bands of 420 bp and 381 bp from Phomopsis sp. However, no band was amplified in the other 10 tobacco pathogen DNAs and the negative controls. The detection sensitivity of both pairs of the specific primers reached 10 pg/μL. The established molecular detection method based on two pairs of specific primers P2F/P3R and P3F/P3R can rapidly and accurately detect Phomopsis sp. in tobacco. |
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