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Codon^(200) TTC→TAC基因型

ASO-PCR amplified by the primers with mismatches to detect moderately carbendazim-resistant genotype (codon^(200) TTC→TAC) of Fusarium asiaticum

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(Codon200TTC→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(Codon200TTC→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。

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.