| 基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法 |
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| 引用本文： | 祝菊澧,梁静思,张佩,王伟伟,林桐司骐,谢欣娱,苏瑞,唐唯.基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法[J].作物杂志,2019,35(6):168-3311. |
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| 作者姓名： | 祝菊澧 梁静思 张佩 王伟伟 林桐司骐 谢欣娱 苏瑞 唐唯 |
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| 作者单位： | 1云南师范大学生命科学学院,650500,云南昆明2云南师范大学马铃薯科学研究院,650500,云南昆明3云南省马铃薯生物学重点实验室,650500,云南昆明 |
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| 基金项目： | 国家自然科学基金(31660503);云南师范大学2019年度研究生科研创新基金(ysdy js2019132) |
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| 摘 要： | 马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 -6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。
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| 关 键 词： | 定量PCR（qPCR） 环介导等温扩增（LAMP） 马铃薯晚疫病菌 潜伏侵染 检测 |
| 收稿时间： | 2019-05-24 |
A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays |
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| Zhu Juli,Liang Jingsi,Zhang Pei,Wang Weiwei,Lin Tongsiqi,Xie Xinyu,Su Rui,Tang Wei.A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays[J].Crops,2019,35(6):168-3311. |
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| Authors: | Zhu Juli Liang Jingsi Zhang Pei Wang Weiwei Lin Tongsiqi Xie Xinyu Su Rui Tang Wei |
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| Institution: | 1School of Life Science, Yunnan Normal University, Kunming 650500, Yunnan, China2Joint Academy of Potato Science, Yunnan Normal University, Kunming 650500, Yunnan, China3Key Laboratory of Potato Biology of Yunnan Province, Kunming 650500, Yunnan, China |
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| Abstract: | Phytophthora infestans can infect many kinds of Solanaceae plants. Potato late blight (PLB) caused by P. infestans is a most severe disease in the potato production. To better and fast diagnose PLB in the fields, quantitative PCR (qPCR) and Loop-mediated isothermal amplification (LAMP) primers were designed based on the P. infestans T30-4 whole genome shotgun contig 1.18131. After amplification conditions optimized, both specificity and sensitivity were detected. Finally, developed qPCR and LAMP methods were used to detect PLB incidence and compared with traditional isolation method on harvested potato tubers in the fields. Specific detection showed that qPCR and LAMP were positive amplification only in the presence of P. infestans DNA template, and were no amplification in the presence of host and other microorganism DNA. Moreover, as low as 1×10 -6ng/μL gDNA was detected using both qPCR and LAMP methods, while the sensitivity of primers showed no significant difference in the presence of P. infestans DNA template, host and other microorganism DNA. Detecting harvested tubers in the field using two methods in Dali, Lijiang and Kunming, qPCR and LAMP showed no significant difference but extremely significant difference between two fast detection methods and morphology based identification. Additionally, in Dali, Lijiang and Kunming, PLB incidence of the two molecular detection methods were both higher than morphology based identification method, in which qPCR detection method was 12.00%, 2.00% and 8.70% higher than morphology based identification method, and LAMP detection method was 11.30%, 2.00% and 8.70% higher than morphology based identification method, respectively. |
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| Keywords: | Quantitative PCR (qPCR) Loop-mediated isothermal amplification (LAMP) Phytophthora infestans Latent infection Detection |
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