双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。

Development and application of duplex PCR assay for detection of Phytophthora infestans and Ralstonia solanacearum

Based on the difference in internal transcribed spacer (ITS) sequences of Phytophthora infestans and other Phytophthora spp. , a specific pair of primers, INF1/INF2, was designed. Among 59 isolates representing seven Oomycetes and 15 other fungi and bacteria species, the primer pair amplified a single 324 bp product from all isolates of P. infestans but not from any other isolates tested. The detection sensitivity increased 1 000-fold to 30 fg genomic DNA by developed a nested PCR procedure with DC6/ITS4 as the first round primers and INF1/INF2 as the second round primers. The duplex PCR system for simultaneous detection of P. infestans and R. solanacearum had been established using the designed combination primers. Under the duplex PCR assay, 324 bp fragment of P. infestans and 281 bp fragment of R. solanacearum could be specifically amplified from the genomic DNA of P. infestans and R. solanacearum. Specificity was confirmed by using the duplex PCR assay to detect P. infestans and R. solanacearum in the natural infected and artificially inoculated potato.