马铃薯晚疫病菌对甲霜灵抗性的快速检测方法

Rapid detection of metalaxyl resistance in potato late blight pathogen Phytophthora infestans

为快速检测马铃薯晚疫病菌——致病疫霉Phytophthora infestans对甲霜灵的抗性,基于已知致病疫霉RPA190基因的T1145A变异引起的F382Y氨基酸点突变与甲霜灵抗性有关,通过设计4对特异性引物F382Y-F1/F382Y-R、F382Y-F2/F382Y-R、F382Y-F3/F382Y-R和F382Y-F4/F382Y-R建立等位基因特异性PCR(allele specific-polymerase chain reaction,AS-PCR)快速分子检测法,其中F382Y-R引物在4对引物里保持不变,并分析所建分子检测法的特异性和灵敏度。结果表明,正向特异性引物F382Y-F2、F382Y-F3和F382Y-F4的3′末端在致病疫霉抗甲霜灵菌株原有核苷酸突变位点1145A(对应引物为F382Y-F1)的基础上,再人工引入第1144位的核苷酸突变,将1144T分别突变成1144G、1144C和1144A,并优化退火温度和特异性引物与内参引物ITS1/ITS4比例,最终确定最适退火温度分别为54、60和58℃,特异性引物与内参引物最适浓度比例均为5∶1。以该3条引物对应的3对特异性引物和内参引物ITS1/ITS4组成的3组多重AS-PCR引物对甲霜灵敏感和抗性菌株具有良好的特异性,敏感菌株的扩增产物含1条879 bp的内参片段,抗性菌株的扩增产物为1条879 bp的内参片段和1条461 bp的目的片段。3组AS-PCR检测体系均具有较高的灵敏度,其中引物F382Y-F4/F382Y-R对致病疫霉DNA的检测灵敏度达0.4 pg/μL,引物F382Y-F2/F382Y-R和F382YF3/F382Y-R的检测灵敏度达4 pg/μL。

In order to detect metalaxyl-resistant strains of potato late blight pathogen Phytophthora infestans rapidly,allele specific-polymerase chain reaction(AS-PCR)methods using four specific primers sets including F382Y-F1/F382Y-R,F382Y-F2/F382Y-R,F382Y-F3/F382Y-R and F382Y-F4/F382Y-R were established based on the known mechanism of metalaxyl resistance associated with amino acid point mutation of F382Y encoded by T1145A in RPA190 gene of P.infestans.The results showed that by using three forward primers F382Y-F2,F382Y-F3 and F382Y-F4,which were designed for point mutation 1145A plus an addition mutations 1144G,1144C and 1144A,respectively,optimizing the annealing temperature to 54,60 and 58℃,respectively,and setting the ratio of the three specific primers to referent primer set ITS1/ITS4 as 5∶1,the three multiple AS-PCRs using specific primers of F382Y-F2/F382Y-R,F382Y-F3/F382Y-R and F382Y-F4/F382Y-R,respectively plus referent primers ITS1/ITS4could distinguish specifically resistant strains from sensitive strains.The amplicons from the sensitive strains contained an internal reference fragment of 879 bp,and the amplicons from the resistant strains showed an internal reference fragment of 879 bp and a target fragment of 461 bp.The three multiple AS-PCR methods all had high sensitivity.Among them,the primers F38Y-F4/F382Y-R had the highest sensitivity in which the minimum detectable concentration of DNA template from P.infestans was0.4 pg/μL,while the sensitivity of F382Y-F2/F382Y-R and F382Y-F3/F382Y-R was both 4 pg/μL.