甘蔗宿根矮化病菌PCR检测技术研究

PCR techniques for detection of Leifsonia xyli subsp.xyli infecting sugarcane

以甘蔗茎组织总DNA为模板,以16S rRNA基因赖氏细菌属(L eif son ia)通用引物为第一轮引物、甘蔗宿根矮化病菌(L eif son ia xy li subsp.xy li,Lxx)亚种特异引物为第二轮引物建立了Lxx巢式PCR检测技术。根据已报道的Lxx巴西分离物基因组全序列(G enB ank登录号AE 016822.1)设计了扩增致病相关基因片段的3个引物对,经多种组合进行了RCR检验,筛选出两个特异性好、灵敏高的引物对,建立了Lxx的多重PCR检测技术。克隆测序表明,PCR产物与巴西分离物基因组相应区段同一率为99%以上,从而证实了上述PCR技术的正确性。检测结果表明,广东样品的阳性率为90%,海南样品的阳性率为60%。

Nest-PCR technique, for detection of sugarcane ratoon stunting disease pathogen Leifsonia xyli subsp, xyli （Lxx）, was established with the total DNA extracted from sugarcane stem as templates, general primers for Leifsonia 16S rDNA as out primers and specific primers for Lxx 16S rDNA as inner primers. A dual PCR technique was also developed with two sets primers selected from three sets primers designed based on reported pathogenic relative genes of Lxx Brazilian isolate （GenBank accession No. AE016822.1）. PCR products were confirmed by cloning and sequencing which showed over 99% nucleotide identities with a Lxx strain from Brazil. Detection results showed that 90% and 60% samples, collected from Guangdong and Hainan Province respectively, were positive.