大豆猝死综合症病原菌北美种的PCR鉴定

 应用等位基因特异性PCR(allele-specific PCR,AS-PCR)技术,建立了大豆猝死综合症(sudden death syndrome of soybean,SDS)病原菌北美种Fusarium virguliforme与其近似种Fusarium phaseoli(菜豆根腐病菌)的鉴别方法。针对翻译延长因子(EF-1α)基因上的3个SNP(单核苷酸多态性)位点,参照引物设计原则和等位基因特异引物(allele-specific primer,ASP)的要求设计了1对引物Fsg-α-1/2,能特异地扩增F.virguliforme,产生327 bp的电泳条带。另外,根据ITS区序列设计了1对通用引物Fu1/2,能扩增F.virguliforme和F.phaseoli,产生452 bp的电泳条带。本实验在传统的AS-PCR基础上进行改进,引入Fu1/2作为阳性质控引物,将ASP的特异性与双重PCR的严谨性相结合,建立了简便可靠的SDS病原菌鉴定方法。

Use of allele-specific PCR of translation elongation factor 1-α gene to identify soy bean sudden death syndrome pathogen (Fusarium virguliforme)

An allele-specific PCR-based method was developed to distinguish Fusarium virguliforme, the causal agent of soybean sudden death syndrome (SDS) in North America, from Fusarium phaseoli. Under the rules of allele-specific primer design, one pair of primers, Fsg-α-1/2 were synthesized based on three SNP sites on translation elongation factor 1-α gene, which could distinctively amplify F.virguliforme and produce PCR products of 327 bp. In addition, to distinguish F.virguliforme and F.phaseoli from the other Fusarium spp., one pair of primers, Fu1/2, were designed based on the region of ITS (intergenic transcribed spacer), and produced PCR products of 452 bp. In this test, conventional AS-PCR and duplex PCR were combined to increase the sensitivity and reproducibility, Fu1/2 acting as positive control primer. Thus, AS-PCR approach provided an accurate, convenient and effective method for quick identification of F.virguliforme.