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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

, PP. 571-577

Keywords: 李属,检疫性真菌,丁香疫霉,栗黑水疫霉,三重PCR检测

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Abstract:

为建立我国禁止进境的2种检疫性真菌丁香疫霉Phytophthorasyringae和栗黑水疫霉P.cambivora的同步分子检测方法,根据疫霉属的18SrRNA、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20s。该体系扩增丁香疫霉出现884bp的18SrRNA条带和683bp的HSP90基因特异条带,扩增栗黑水疫霉出现884bp的18SrRNA条带和314bp的Ypt1基因特异条带,对照菌只出现18SrRNA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。

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