马铃薯黑痣病多重实时定量 PCR 检测方法的建立

摘要

The TaqMan RFQ-PCR multiple real-time quantification detection method was established to detect conserved sequence-Internal transcribed spacer (ITS)and Cox gene of Rhizoctonia solani Kn rDNA gene cluster of potato black scurf pathogenic bacteria simultaneously.The results showed that the new RFQ-PCR multiple real-time quantification detection method with advantages of strong specificity, high sensitivity,simplicity,rapidity and reliability can be used in detection of Rhizoctonia solani of potato black scurf because its primer-probe is of higher specificity and higher sensitivity,and introduction of reference gene can avoid appearance of false negative effectively.%为建立快速、准确、可靠的马铃薯黑痣病鉴定和检测方法，以黑痣病病原菌立枯丝核菌核糖体基因簇间的保守序列—转录间隔区（Internal transcribed spacer，ITS）为靶区域，同时引入马铃薯细胞色素氧化酶（cytochrome oxidase gene，Cox）作内参，应用多重实时定量 PCR 技术，建立马铃薯黑痣病病原菌TaqMan RFQ-PCR 多重快速检测方法。结果表明：RFQ-PCR 检测体系的引物-探针具有高度的特异性，灵敏度比普通 PCR 方法高10～100倍；内参基因的引入还可有效避免假阴性的出现。方法特异性强、灵敏度高、简单、快速、可靠。