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由荔枝霜疫霉(Peronophythora litchii)引起的荔枝霜疫霉病是荔枝(Litchi chinensis)生产上的重要病害,建立霜疫霉快速准确检测方法,对于该病的早期诊断和及时防控至关重要。本研究以荔枝霜疫霉三磷酸鸟苷(GTP)结合蛋白基因(GTP-binding protein gene,Ypt1)为靶序列,设计特异性引物,建立了巢式PCR和环介导等温扩增(loop-mediated isothermal amplification,LAMP)两种检测方法,并进行特异性和灵敏度验证。特异性检测表明,只有不同来源的21个荔枝霜疫霉菌株经PCR扩增获得249 bp的特异性条带,同时,在钙黄绿素指示剂的作用下,LAMP检测显示绿色,且扩增产物用2.0%琼脂糖凝胶电泳出现特有的梯形条带,而其他13种不同卵菌近缘种及8种常见病原真菌42个菌株均未观察到这些现象。灵敏度检测显示,将特异引物Pv YF1/Pv YR1与疫霉属Ypt1通用引物Yph1F/Yph2R进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达100 fg/25μL,较常规PCR提高1 000倍,而LAMP方法检测灵敏度是巢式PCR的10倍。本研究分别采用常规PCR、巢式PCR和LAMP方法对采集自福建省荔枝霜疫霉病典型症状及可疑症状的荔枝叶片或果实进行检测,并用传统分离方法进行验证。结果表明,在漳州采集的30份样品中,常规PCR、巢式PCR和LAMP方法的阳性检出率分别为17/22(77.3%)、20/22(90.9%)和21/22(95.5%);在莆田采集的25份样品中,常规PCR、巢式PCR和LAMP方法的阳性检出率分别为15/17(88.2%)、16/17(94.1%)、17/17(100%)。可见LAMP方法明显提高了检测效率,并且具有检测程序便捷,所需设备简单和肉眼能判断结果的优势,适合基层部门及田间荔枝霜疫霉快速检测。
Phytophthora licheniformis caused by Peronophythora litchii is an important disease in the production of Litchi chinensis. It is very important to establish a rapid and accurate method for the detection of Phytophthora capsici in the early diagnosis and prevention of the disease . In this study, specific primers were designed based on the target sequence of GTP-binding protein gene (GTP-binding protein gene, Ypt1). Nested PCR and loop-mediated isothermal amplification- mediated isothermal amplification (LAMP), and to verify the specificity and sensitivity. Specificity tests showed that only 249 bp specific bands were obtained by PCR amplification from 21 P. lycoophylla strains of different origins and the LAMP assay showed green color under the action of calcein indicator, 2.0% agarose gel electrophoresis showed unique trapezoidal bands, and other 13 kinds of different Oomycetes relatives and 8 kinds of common pathogenic fungi 42 strains were not observed in these phenomena. Sensitivity analysis showed that the detection sensitivity of Pv YF1 / Pv YR1 and Yph1F / Yph2R was 100 fg / 25μL at the DNA level, which was 1 000 higher than that of the conventional PCR Times, and LAMP method detection sensitivity is 10 times that of nested PCR. In this study, the conventional PCR, nested PCR and LAMP methods were used to detect the litchi leaves or fruits collected from the typical symptoms and suspicious symptoms of frost and fungus disease in Fujian Province, respectively. The results were verified by traditional methods. The results showed that the positive detection rates of routine PCR, nested PCR and LAMP were 17/22 (77.3%), 20/22 (90.9%) and 21/22 (95.5%) in 30 samples collected in Zhangzhou, ). The positive detection rates of routine PCR, nested PCR and LAMP in 25 samples collected in Putian were 15/17 (88.2%), 16/17 (94.1%) and 17/17 (100%), respectively . It can be seen that the LAMP method obviously improves the detection efficiency, and has the advantages of convenient detection procedures, simple equipment and naked eyes to judge the results, and is suitable for the rapid detection of Phytophthora Licheni in grass-roots units and in the field.