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

 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。

ASO-PCR amplified by the primers with mismatches to detect moderately carbendazim-resistant genotype (codon200TTC→TAC) of Fusarium asiaticum

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.