利用错配碱基引物的ASO-PCR检测小麦赤霉病菌对 多菌灵中抗菌株Codon200 TTC→TAC基因型 |
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引用本文: | 陈长军,蔡倩,王建新,周明国.利用错配碱基引物的ASO-PCR检测小麦赤霉病菌对 多菌灵中抗菌株Codon200 TTC→TAC基因型[J].植物病理学报,2011,41(3):278-284. |
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作者姓名: | 陈长军 蔡倩 王建新 周明国 |
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基金项目: | 国家“973”项目 (2006CB101900); 国家/江苏省自然科学基金项目(30671048,30671348)/BK2008337; 国家博士点基金项目(20050307034) |
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摘 要: | 采用在引物3′端引入错配碱基,建立了特异性检测小麦赤霉病菌对多菌灵中抗菌株(Codon200 TTC→TAC)基因型的ASO-PCR分子检测技术。结果表明含有错配碱基的引物对NT-7 R1/NT-7 Err5 F能够特异性检测小麦赤霉病菌对多菌灵的中抗菌株(Codon200 TTC→TAC),扩增条件为94℃预热5 min;94℃变性60 S,56℃退火60 S,72℃延伸60 S, 35个循环;最后72℃延伸15 min。并利用26种常见植物病原真菌验证了所设计引物的PCR扩增特异性。整个检测过程快速,操作简单,结果准确,在6小时内完成。
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关 键 词: | 小麦赤霉病菌 多菌灵抗药性 ASO-PCR检测 Codon200 TTC&rarr TAC基因型 错配碱基引物 |
收稿时间: | 2010-01-18; |
ASO-PCR amplified by the primers with mismatches to detect moderately carbendazim-resistant genotype (codon200TTC→TAC) of Fusarium asiaticum |
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Abstract: | An allele specific oligonucleotide-PCR (ASO-PCR) with mismatches at the 3′ terminal of the primer was set up to detect moderately carbendazim (MBC)-resistant genotype (Codon200 TTC→TAC) of Fusarium asiaticum. The results showed that a pair of probe, NT-7 R1/NT-7 Err5 F with mismatches was deve-loped to successfully detect moderately MBC-resistant isolates (Codon200 TTC→TAC). The ASO-PCR amplification by MBCRF/MBCRR3 was conducted with the following parameters: an initial pre-heat at 94℃ for 5 min, following by 35 cycles of denaturation at 94℃ for 60 s, annealing at 56℃ for 60 s, extension at 72℃ for 60 s and terminated with a final extension at 72℃ for 15 min. 26 important plant fungi were used to test the amplification specificity of the primer pair. This method is simple,accurate and time-saving.The result was obtained within 6 h. |
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Keywords: | Fusarium asiaticum Fusarium asiaticum resistance of MBC allele specific oligonucleotides-PCR with mismatches genotype of Codon rarr TAC mismatch primers |
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