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为建立我国禁止进境的2种检疫性真菌丁香疫霉Phytophthora syringae和栗黑水疫霉P.cambivora的同步分子检测方法,根据疫霉属的18S rRNA、HSP90和Ypt1基因分别设计通用引物、丁香疫霉和栗黑水疫霉的特异性引物,建立三重PCR检测方法,并进行灵敏度测试和模拟带菌试验。结果表明,可同时检测李属植物上丁香疫霉和栗黑水疫霉的特异三重PCR检测体系为:最佳引物浓度组合18SUF/18SUR、PCSF/PCSR和PSSF/PSSR依次为0.2、0.8、1.0μL,最佳退火温度为63℃,最佳退火时间为20 s。该体系扩增丁香疫霉出现884 bp的18S rRNA条带和683 bp的HSP90基因特异条带,扩增栗黑水疫霉出现884 bp的18S rRNA条带和314 bp的Ypt1基因特异条带,对照菌只出现18S rRNA条带;三重PCR反应体系检测灵敏度低于单重PCR;模拟带菌试验可同时扩增出3个片段。表明该三重PCR检测方法能实现丁香疫霉和栗黑水疫霉的同步特异性检测,可有效改进李属类水果及其种苗上检疫性疫霉的快速检测。
In order to establish a synchronous molecular detection method of Phytophthora syringae and P. cambivora, two quarantine fungi that are forbidden to enter China, universal primers were designed according to 18S rRNA, HSP90 and Ypt1 genes of Phytophthora, Mold and chestnut black Phytophthora infestans specific primers, the establishment of triplex PCR detection method, and the sensitivity test and simulated carriage test. The results showed that the specific triplex PCR system for simultaneous detection of Phylla spp. And P. plutella could be described as follows: the best primer concentration combination 18SUF / 18SUR, PCSF / PCSR and PSSF / PSSR were 0.2,0.8,1.0μL , The best annealing temperature is 63 ℃, the best annealing time is 20 s. The system amplified 884 bp of 18S rRNA and 683 bp of HSP90 specific bands, amplified 884 bp of 18S rRNA and 314 bp of Ypt1 specific bands, Only 18S rRNA bands appeared in the control strain. The detection sensitivity of the triplex PCR reaction system was lower than that of the single PCR. The mimic banding test could amplify 3 fragments simultaneously. The results showed that the triplex PCR method could detect the synchronous specificity of Phytophthora capsici and Phthisidium pluvialis, which could effectively improve the rapid detection of quarantine Phytophthora on Prunus fruits and their seedlings.