作　　者： ; ; ;

机构地区： 华东理工大学生物工程学院生物反应器工程国家重点实验室

出　　处： 《中国农业科学》 2006年第10期2028-2035,共8页

摘　　要： 【目的】探索西瓜炭疽病菌(Colletotrichum orbiculare)的分子检测技术,为西瓜、甜瓜等生长期和采后炭疽病的快速、准确鉴定和检测提供技术和方法。【方法】根据GeneBank中Colletotrichum属的24个种ITS序列,比较设计出1对引物CY1/CY2(CY1:5′-CTTTGTGAACATACCTAACC-3′;CY2:5′-GGTTTTACGGCAGGAGTG-3′);进一步利用RAPD随机引物扩增西瓜炭疽菌C.orbiculare和菜豆炭疽菌C.lindemuthianum,找出在C.orbiculare中的特异性条带,经过克隆、测序后,设计出1对SCAR引物RB/RC。【结果】引物CY1/CY2可以特异的从C.orbiculare和C.lindemuthianum菌株中扩增到1条442 bp的条带,将这2个种的炭疽菌和其它炭疽菌种以及其它真菌种分开。引物RB/RC可以特异地在C.orbiculare中扩增出1条216 bp条带,将C.orbiculare和C.lindemuthianum分开。采用2对引物组成双重PCR,将C.orbiculare进行特异性扩增,可获得442 bp和216 bp的2条特异性条带。【结论】利用上述2对引物组成双重PCR检测体系,快速鉴定C.orbiculare,并且能够直接在植物组织中将西瓜炭疽菌C.orbiculare检测出来,灵敏度可以达到1pg.μl-1。 [Objective] A duplex PCR was developed to detect Colletotrichum orbiculare, a causal agent of watermelon anthracnose, in infectedd leaves and fruits. [ Method] Based on the differences of ITS DNA sequences of 24 species of Colletotrichum from GenBank, a couple of specific primers of CY1/CY2 （CYl： 5＇-CTTTGTGAACATACCTAACC-3＇; CY2： 5＇-GGTITFACGGCAGGAGTG-3＇） were synthesized. By RAPD technique, a band from C. orbiculare was specific amplified, and then the band was cloned, sequenced and used to design specific primers of RB/RC （RB： 5＇-GCTGTCACTTTGTGGTGTG-3＇; RC： 5＇-TGTCGTAGCCCATCTTGTC-3＇） for this species. [Result] Using CY1/CY2 primers, only a single PCR band of 442 bp from C. orbiculare and C. lindemuthianum was amplified and no PCR band from other species. The RB/RC primers could identify and distinguish C. orbiculare from C. lindemuthianum the 216 bp PCR band. A duplex PCR method, combining primers CYI/CY2 and RB/RC, was used to detect C. orbiculare. The detection sensitivity with the two couples of primers was 1pg·μl^-1 of genomic DNA. [Conclusion] The PCR-based method could be used for the accurate and rapid identification of C. orbiculareon watermelon in the growth stage and postharvest.

关 键 词： 西瓜炭疽病 分子检测 双重

领　　域： [农业科学] [农业科学]