李属植物检疫性丁香疫霉和栗黑水疫霉的三重PCR分子检测

Triplex-PCR molecular detection of two quarantine fungal diseases of Prunus spp.,Phytophthora syringae and P. cambivora

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 for the two China entryforbidden quarantine fungal pathogens of Prunus spp.,Phytophthora syringae and P. cambivora,the universal primers 18 SUF /18 SUR and the specific primers PSSF / PSSR for P. syringae and PCSF / PCSR for P. cambivora were designed based on the 18 S rRNA,HSP90 and Ypt1 genes,respectively,to establish a triplex-PCR detection and carry out its sensitivity test and simulating carrier detection. The established specific triplex PCR reaction system that could simultaneously detect P. syringae and P.cambivora of Prunus was as follows： the optimal primers combination of 18 SUF /18 SUR,PCSF / PCSR and PSSF / PSSR was 0. 2,0. 8,and 1. 0 μL,respectively,and the optimal annealing temperature and time were 63℃ and 20 s,respectively. Application detected an 884 bp band of 18 S rRNA and a 683 bp specific band to the HSP90 gene in P. syringae,and an 884 bp band of the 18 S rRNA and a 314 bp specific band of the Ypt1 gene were also detected in P. cambivora,while only a band of 18 S rRNA wasdetected in the control. The sensitivity of the triplex-PCR was lower than that of conventional PCR. The simulated carrier detection detected the three bands simultaneously. The established triplex-PCR could achieve synchronous and specific detection of P. syringae and P. cambivora, and thus could be effectively used to promote quick detection of Prunus fruits and its seedlings.