检疫性柑橘冬生疫霉和丁香疫霉的三重 PCR 同步检测

全世界危害柑橘的疫霉有14种,其中冬生疫霉Phytophthora hibernalis和丁香疫霉Phytophthora syringae为我国规定禁止进境的两种检疫性柑橘真菌病害,目前尚未见有此两种病原菌的同步分子检测报道.该研究根据14种柑橘疫霉的18SrRNA,ITS,heat shock protein 90(HSP 90)等基因分别设计了疫霉属的通用引物和此两种检疫性疫霉的特异引物,通过进行反应体系优化,建立了同时检测柑橘上两种检疫性疫霉的特异三重PCR检测方法,并进行了灵敏度试验.用15个柑橘疫霉菌株与甜橙的混合DNA做模板进行模拟带菌试验,结果表明该三重PCR分子检测能实现两种检疫性疫霉菌的同步特异检测,这两种检疫性柑橘疫霉提供了特异、可靠、便捷、适于口岸应用的检测方法,可有效促进柑橘类水果的快速通关.

Simultaneous Triplex-PCR Detection of Two Quarantine Fungal Pathogens of Citrus :Phy tophthora hibernalis and Phy tophthora sy ringae

There are 14 Phytophthora pathogen species in citrus across the world ,of which P. hibernalis and P. syringae are prohibited to enter China .So far no simultaneous molecular detection of the two pathogens has been reported .In the present study ,universal primers for the genus Phytophthora and two pairs of specific primers for the two quarantine Phytophthora species were designed based on the 18S rRNA ,ITS and heat shock protein 90 gene (HSP 90) sequences of the 14 Phytophthora pathogens . Through the optimization of the reaction conditions ,a specific and simultaneous triplex detection method (riplex PCR) for P. hibernalis and P. syringae on citrus was established ,and its sensitivity was tested . The simulated carrier detection by the mixed DNA template of all the 15 citrus Phytophthora strains and the sweet orange proved that P. hibernalis and P. syringae can be detected by the triplex PCR simultane-ously and specifically .Thus ,the established triplex PCR is a specific and reliable method to detect the two quarantine fungal diseases ,suitable for surveillance at frontier ports ,and can be used effectively to pro-mote the customs clearance of citrus fruits .