基于环介导等温扩增技术快速检测瓜果腐霉菌

[目的]建立一种快速、准确的检测瓜果腐霉菌（Pythium aphanidermatum）的环介导等温扩增（loop-mediated isothermal amplification，LAMP）方法，为诊断和防治瓜果腐霉菌引起的植物病害提供技术支撑。[方法]通过比较基因组学方法，在瓜果腐霉菌基因组上筛选有序列特异性的基因作为检测的靶标基因，设计LAMP特异性引物，建立一种基于颜色判定的快速、准确的LAMP检测方法，随后对该检测方法的特异性、灵敏度和发病组织实际检测等进行分析评价。[结果]鉴定到1个编码Trypsin蛋白酶的基因是瓜果腐霉菌特异的基因，以该序列作为靶标建立了LAMP检测方法。在扩增前加入染料羟基萘酚蓝（HNB）作为指示剂，在等温条件（64℃）下进行核酸扩增反应60 min，即可通过肉眼直接观察结果，HNB显示天蓝色即为阳性反应；同时，扩增产物经琼脂糖凝胶电泳验证为梯形条带，也可判定为阳性反应。特异性试验中，建立的LAMP检测方法能将瓜果腐霉菌和其他病原菌区分开。灵敏度试验中，LAMP检测方法的最低检测限为100 pg？μL-1，与Real-time PCR反应灵敏度一样，是普通PCR反应灵敏度的100倍。发病组织实际检测试验中，该LAMP方法能检测出人工接种发病植株中的瓜果腐霉菌。[结论]成功建立了一种适用于基层使用的针对瓜果腐霉菌的快速检测技术，为该菌所致病害的准确诊断奠定了技术基础。
[Objectives]The objective of this study is to establish a rapid and accurate loop-mediated isothermal amplification(LAMP) method for detecting Pythium aphanidermatum,and to provide technical supports for monitoring and prevention of plant diseases caused by P.aphanidermatum. [Methods]A comparative genomics approach was used to identify specific target gene for P.aphanidermatum. The LAMP method was established,and was evaluated for specificity,sensitivity,and detection of inoculated plant tissues. [Results]One gene encoding Trypsin protease was specific for P.aphanidermatum,and was used as the target. The LAMP method efficiently amplified the target gene at 64℃ for 60 min,and LAMP products were visualized by adding hydroxynaphthol blue(HNB) before amplification. The amplification products that turned blue were considered to be positive. Meanwhile,the positive DNA products were visualised as a ladder-like banding pattern on 20 g？L-1 gel electrophoresis. The specificity test showed that positive results were only obtained from P.aphanidermatum isolates. The detection limit was 100 pg？μL-1,which was the same with Real-time PCR,and was nearly 100 times higher than that of the conventional PCR. In addition,this LAMP method was successfully applied to detect P.aphanidermatum DNA extracted from inoculated plant tissues. [Conclusions]We successfully established a simple,rapid and accurate LAMP method for detecting P.aphanidermatum in the field