稻曲病菌的SCAR标记及其PCR检测

SCAR marker and PCR detection of Ustilaginoidea virens

为建立稻曲病菌Ustilaginoidea virens快速、灵敏的PCR早期检测技术,以S380为引物,对稻曲病菌和其它参试病原菌的DNA进行RAPD-PCR扩增。稻曲病菌RAPD特异片段经回收、克隆和测序后,根据序列用Primer 5.0软件设计了特异性SCAR引物US-SF(5'-TCGCCTCAGACCTCAATC-3')/US-SR(5'-GAGCCTCAAATGCCTTCC-3')和巢式PCR引物US-NF(5'-AGCGTCTCCTGCAACCAC-3')/US-NR(5'-GAGCCTCAAATGCCTTCC-3')。利用引物US-SF/US-SR通过常规PCR对供试稻曲病菌均可扩增出1条大小约257 bp的清晰条带,对其它植物病原菌DNA的PCR产物均无扩增条带,最低可检测到1 pg/μL的病菌基因组DNA;而利用引物US-SF/US-SR和US-NF/US-NR通过巢式PCR可从10 fg/μL的病菌基因组DNA中扩增出1条大小约210 bp的特异性条带。试验表明,巢式PCR检测灵敏度比常规PCR提高了100倍,且巢式PCR可从接菌的水稻幼颖和自然感染的谷粒中检测出稻曲病菌。

To develop a rapid and sensitive technique for early PCR detection of Ustilaginoidea virens in plants, the primers of S380 were used to amplify DNA of U.virens isolates and other pathogen isolates tested by RAPD-PCR. The specific RAPD fragment of U.virens was collected, cloned and sequenced, based on the sequence of RAPD marker; the specific SCAR primers US-SF（5＇-TCGCCTCAGACCTCAATC-3＇）/US-SR（5＇-GAGCCTCAAATGCCTTCC-3＇）and nested PCR primers US-NF（5＇-AGCGTC-TCCTGCAACCAC-3＇）/US-NR（5＇-GAGCCTCAAATGCCTTCC-3＇） were designed by the software Primer 5.0. The conventional PCR product of all tested isolates of U.virens showed a clear and bright fragment of about 257 bp with the SCAR primers US-SF/US-SR, but other pathogen had no product amplified. The minimal genomic DNA to be detected reached to 1 pg/μL, but nested PCR using the primers US-SF/US-SR and US-NF/US-NR could detect the specific fragment of about 210 bp from 10 fg/μL of genomic DNA. The results showed that detection sensitivity of the nested PCR was increased by 100-fold compared to the conventional PCR, and the U.virens from rice glumes inoculated and grains infected naturally could be detected by the nested PCR.