摘要
麦类壳多胞斑点病菌(Parastagonospora pseudonodorum)、小麦基腐病菌(Oculimacula yallundae)、小麦全蚀病菌(Gaeumannomyces graminis var.tritici)是麦类作物上的重要检疫性病原真菌。建立一种可以同时检测上述3种植物病原真菌的三重荧光PCR方法,可以实现进境麦类粮食中这3种病原真菌的快速准确检疫鉴定。在对麦类壳多胞斑点病菌TUB2基因、小麦基腐病菌ITS1基因、小麦全蚀病菌EF1-α基因单重荧光PCR扩增引物与探针浓度优化的基础上,对三重荧光PCR扩增的引物与探针浓度进行优化,据此进一步对三重荧光PCR扩增程序的退火温度进行优化,建立了可同时检测上述3种植物病原真菌的三重荧光PCR方法,并对该方法的特异性和灵敏度分别进行了验证。结果表明:三重荧光PCR扩增时,TUB2基因、ITS1基因、EF1-α基因的引物与探针最优添加量分别为0.5、0.6和0.6μL,最优退火温度为60℃。特异性验证结果显示,3种靶标菌株均出现了显著扩增;而10株非靶标菌株均未出现扩增,结果为阴性。灵敏度验证结果显示,3种靶标病菌的检测灵敏度分别为41、39、45 fg/μL。本研究建立的三重实时荧光PCR检测方法特异性强、灵敏度高,能够同时快速准确鉴定麦类壳多胞斑点病菌、小麦基腐病菌、小麦全蚀病菌,适用于口岸进境粮食这3种病原真菌的快速准确鉴定。
Parastagonospora pseudonodorum,Oculimacula yallundae and Gaeumannomyces graminis var.tritici are important quarantine pathogens in wheat crops.Establishing a triple fluorescent PCR method that can simultaneously 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 concentration of primers and probes for single fluorescence PCR amplification of the P.pseudonodorum TUB2 gene,O.yallundae ITS1 gene and G.graminis var.tritici EF1-琢gene,the concentration of primers and probes for triple fluorescent PCR amplification was optimized.Based on this,the annealing temperature of the triple fluorescent 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 optimal addition amounts of primers and probes for TUB2 gene,ITS1 gene,and EF1-琢gene were 0.5,0.6,and 0.6滋L,respectively,and the optimal annealing temperature was 60益.The specificity verification results showed that all three target strains showed significant 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/滋L 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.
作者
李金庆
季蕾
郭骁驹
邵秀玲
杨斌
孙民琴
张静
王云霞
LI Jin-qing;JI Lei;GUO Xiao-ju;SHAO Xiu-ling;YANG Bin;SUN Min-qin;ZHANG Jing;WANG Yun-xia(Technical Center of Qingdao Customs,Qingdao 266109,China;Technical Center of Yantai Customs,Yantai 264000,China;Nantong Customs,Nantong 226001,China)
出处
《河北农业科学》
2024年第4期99-108,共10页
Journal of Hebei Agricultural Sciences
基金
国家重点研发计划项目(2021YFD1400100,2021YFD1400103)
青岛海关科研项目(QK202053)。
关键词
三重荧光PCR
麦类壳多胞斑点病菌
小麦基腐病菌
小麦全蚀病菌
快速检测
Triple fluorescent PCR
Parastagonospora pseudonodorum
Oculimacula yallundae
Gaeumanno原myces graminis var.tritici
Detection
