Xiong Qin, Zhang Linlin, Zheng Xinyue, Qian Yulin, Zhang Yaxin, Zhao Lijuan, Cheng Qiang
Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China.
The Southern Modern Forestry Collaborative Innovation Center, Nanjing Forestry University, Nanjing 210037, China.
Plants (Basel). 2021 Jan 28;10(2):253. doi: 10.3390/plants10020253.
is the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists for . Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of . A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer () region of . The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA of changed from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two other pathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates of from artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, the LAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection of in poplar endemic areas with resource-limited settings.
是引起杨树黑斑病的主要病原菌，导致光合效率下降，严重影响木材产量和质量。目前，尚无针对的田间诊断方法。在此，我们描述了一种环介导等温扩增（LAMP）检测方法，用于快速灵敏地检测。设计了一组六条寡核苷酸引物，以识别的内部转录间隔区（）的八个不同序列。通过结合高特异性、灵敏度和快速性对LAMP检测进行了优化，可在65℃下每反应60分钟检测低至10 pg/μL的目标基因组DNA，而PCR检测浓度低于1 ng/μL的DNA时无扩增。在20种真菌分离物的基因组DNA中，仅含有基因组DNA的样品使用羟基萘酚蓝（HNB）染料后从紫色变为天蓝色（肉眼可见）。从其他八种真菌中未扩增出DNA，包括另外两种病原菌、杨树的另外三种叶部真菌病原菌以及杨树的三种常见叶部真菌内生菌。此外，使用PCR检测人工和自然感染的杨树叶片中的检出率分别为10/16（62.5%）和6/16（37.5%），而使用LAMP检测的阳性样本率均为16/16（100%）。总体而言，本文建立的LAMP检测方法可作为基于PCR技术的更好替代方法，用于在资源有限的杨树流行地区特异性灵敏地检测。
