Establishment of Triple Fluorescence PCR Detection Method for Three Quarantine Pathogenic Fungi in Wheat Grains
Parastagonospora pseudonodorum,Oculimacula yallundae and Gaeumannomyces graminis var.tritici are important quarantine pathogens in wheat crops.Establishing a triple fluorescent PCR method that can simul-taneously detect the three plant pathogenic fungi mentioned above can achieve rapid and accurate quarantine and identification of these three pathogenic fungi on imported wheat grains.On the basis of optimizing the con-centration of primers and probes for single fluorescence PCR amplification of the P.pseudonodorum TUB2 gene,O.yallundae ITS1 gene and G.graminis var.tritici EFl-a gene,the concentration of primers and probes for triple fluorescent PCR amplification was optimized.Based on this,the annealing temperature of the triple fluo-rescent PCR amplification was further optimized,and a triple fluorescent PCR method capable of simultaneously detecting the three plant pathogenic fungi mentioned above was established,and the specificity and sensitivity of the method were verified separately.The results showed that during triple fluorescent PCR amplification,the op-timal addition amounts of primers and probes for TUB2 gene,ITS1 gene,and EF1-a gene were 0.5,0.6,and 0.6 μL,respectively,and the optimal annealing temperature was 60 ℃.The specificity verification re-sults showed that all three target strains showed sig-nificant amplification,while 10 non target strains did not show amplification,resulting in a negative result.The sensitivity verification results showed that the de tection sensitivity of the three pathogens was 41,39 and 45 fg/μL respectively.In summary,the triple real-time fluorescent PCR detection method established in the study has strong specificity and high sensitivity,and can quickly and accurately identify the three pathogens of P.pseudonodorum,O.yallundae and G.graminis var.tritici simultaneously.It is suitable for the rapid and accurate identification of these three pathogens in imported grains at ports.
万方数据
维普
