苹果属3种检疫性疫霉的四重PCR分子检测
发布时间：2019-05-21 18:19
【摘要】：为建立苹果属冬生疫霉Phytophthora hibernali、丁香疫霉P.syringae和栗黑水疫霉P.cambivora 3种检疫性疫霉的同步分子检测方法,根据疫霉属18S rRNA,ITS,HSP90和Ypt1基因分别设计通用引物,冬生疫霉、丁香疫霉及栗黑水疫霉特异引物,建立了四重PCR检测方法,并进行了灵敏度和模拟带菌测试.建立了可同时检测苹果属上冬生疫霉、丁香疫霉和栗黑水疫霉的特异四重PCR检测体系:在20μL反应体系中,最佳引物浓度组合为10μmol/L的18SUF/18SUR,PHSF/PHSR,PCSF/PCSR和PSSF/PSSR分别为0.2,0.6,0.8,1.0μL;最佳退火温度和退火时间分别为63℃和20s.该体系扩增冬生疫霉出现884bp的18S rRNA条带和232bp的ITS基因特异带,丁香疫霉出现884bp的18S rRNA条带和683bp的HSP90基因特异带,扩增栗黑水疫霉出现884bp的18S rRNA条带和314bp的Ypt1基因特异带,对照菌只出现18S rRNA条带;四重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4个片段.表明该四重PCR检测方法能实现冬生疫霉、丁香疫霉和栗黑水疫霉的同步特异检测,实现苹果属类水果及种苗检疫性疫霉的快速检测.
[Abstract]:In order to establish a synchronous molecular method for the detection of three kinds of quarantine Phytophthora, Phytophthora hibernali, clove P.syringae and P.cambivora, according to the 18s rRNA,ITS,HSP90 and Ypt1 genes of Phytophthora, general primers were designed for the detection of Phytophthora. A four-fold PCR method was established for the detection of Phytophthora clove and Phytophthora mollissima with specific primers, and the sensitivity and simulated carrying test were carried out. A specific quadruple PCR system for the simultaneous detection of Phytophthora, Phytophthora clove and Phytophthora mollissima was established. In the 20 渭 L reaction system, the optimum primer concentration was 18SUF 鈮，
本文编号：2482291
[Abstract]:In order to establish a synchronous molecular method for the detection of three kinds of quarantine Phytophthora, Phytophthora hibernali, clove P.syringae and P.cambivora, according to the 18s rRNA,ITS,HSP90 and Ypt1 genes of Phytophthora, general primers were designed for the detection of Phytophthora. A four-fold PCR method was established for the detection of Phytophthora clove and Phytophthora mollissima with specific primers, and the sensitivity and simulated carrying test were carried out. A specific quadruple PCR system for the simultaneous detection of Phytophthora, Phytophthora clove and Phytophthora mollissima was established. In the 20 渭 L reaction system, the optimum primer concentration was 18SUF 鈮，
本文编号：2482291
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