基于微滴式数字PCR技术多重精准检测鉴定三种常见检疫性粉蚧OA
Droplet digital PCR for the multiplex and precise identification of three common quarantine mealybug species(Hemiptera:Pseudococcidae)
[目的]应用微滴式数字PCR(droplet digital PCR,ddPCR)技术,建立大洋臀纹粉蚧Pianococcus minor、南洋臀纹粉蚧Pl.lilacinus和木瓜秀粉蚧Paracoccus marginatus 3种高频截获检疫性粉蚧多重精准检测方法.[方法]以口岸截获和国内采集的常见粉蚧大洋臀纹粉蚧、南洋臀纹粉蚧、木瓜秀粉蚧、新菠萝灰粉蚧Dysmicoccus neobrevipes、杰克贝尔氏粉蚧Pseudococcus jackbeardsleyi、木槿曼粉蚧Maconellicoccus hirsutus、康氏粉蚧Ps.comstocki和桔臀纹蚧粉Pl.citri为参试材料,选择CO I基因序列为靶标,使用GenBank数据库、MEGA11和Beacon Designer 8等进行同源性比对和特异性引物与探针设计;在构建的大洋臀纹粉蚧、南洋臀纹粉蚧和木瓜秀粉蚧3种粉蚧单重ddPCR检测体系的基础上,通过筛选最佳引物和探针浓度优化反应体系,建立可同时快速、绝对定量检测大洋臀纹粉蚧、南洋臀纹粉蚧和木瓜秀粉蚧基因组拷贝数的多重ddPCR检测方法,并与TaqMan qPCR方法进行比较,测定该方法的灵敏度、定量限及准确性.[结果]构建的多重ddPCR检测体系内各组引物和探针对目标粉蚧基因组均能扩增,未出现交叉反应,DNA最低检测浓度与TaqMan qPCR中的一致.这一多重ddPCR体系对大洋臀纹粉蚧基因组的绝对定量检测低限为0.44拷贝/μL,对南洋臀纹粉蚧基因组的绝对定量检测低限为0.43拷贝/μL,对木瓜秀粉蚧基因组的绝对定量检测低限为0.44拷贝/μL.在定量检测范围内这一多重ddPCR体系定量检测的线性决定系数R2均大于0.99,且重复性和线性相关性较好.[结论]本研究建立的多重ddPCR检测方法可实现对3种高频截获检疫性粉蚧大洋臀纹粉蚧、南洋臀纹粉蚧和木瓜秀粉蚧快速、精准鉴定,为口岸外来有害生物防控和保护我国生态安全提供技术服务和支持.
[Aim]To apply the droplet digital PCR(ddPCR)technology to establish a multiplex and precise detection method for three quarantine mealybug species,Planococcus minor,Pl.lilacinus and Paracoccus marginatus intercepted at high frequency.[Methods]Using common mealybug species Pl.minor,Pl.lilacinu,Pa.marginatus,Dysmicoccus neobrevipes,Pseudococcus jackbeardsleyi,Maconellicoccus hirsutus,Ps.comstocki and Pl.citri intercepted at ports and collected domestically as test insects,the CO I gene sequence was used as the target.Homology alignment and design of specific primers and probes were performed using the GenBank database,MEGA11 and Beacon Designer 8.Based on the single ddPCR detection system of the three mealybug species Pl.minor,Pl.lilacinus and Pa.marginatus,the reaction system was optimized by screening the optimal concentrations of primers and probes,thereby establishing a multiplex ddPCR method for the rapid and absolute quantitative detection of the genomic copy numbers of Pl.minor,Pl.lilacinus and Pa.marginatus in the same reaction system,and compared with TaqMan qPCR method to determine the sensitivity,quantification limit and accuracy of this method.[Results]All sets of primers and probes could amplify the genomes of target mealybugs in the multiplex ddPCR detection system without cross-reactivity,and the lowest DNA concentration detected was consistent with that of TaqMan qPCR.The lower limit of absolute quantification of this nultiplex ddPCR detection system for the genomic detection of Pl.minor was 0.44 copies/μL,that for the genomic detection of Pl.lilacinus was 0.43 copies/μL,and that for the genomic detection of Pa.marginatus was 0.44 copies/μL.The linear coefficients of determination R2 for the quantitative assay of this nultiplex ddPCR detection system were all greater than 0.99 within the range of quantification,and the reproducibility and linear correlation were good.[Conclusion]The multiplex ddPCR detection method established in this study can realize the rapid and precise identification of three quarantine mealybug species Pl.minor,Pl.lilacinus and Pa.marginatus intercepted at high frequency,and provide technical service and support for the prevention and control of foreign pests at ports and the protection of China's ecological security.
李鑫;李辉;李立梅;杨力凤;孟凡泽;褚栋;付海滨
大连海关技术中心,大连 116000东港海关综合技术服务中心,丹东 118300吉林省林业科学研究院,长春 130000青岛农业大学植物医学学院,青岛 266109University of Minnesoda,Twin Cities,Twin 55455,Unites States of America青岛农业大学植物医学学院,青岛 266109沈阳海关技术中心,沈阳 110000
生物科学
大洋臀纹粉蚧南洋臀纹粉蚧木瓜秀粉蚧分子鉴定分子标记微滴式数字PCR
Planococcus minorPlanococcus lilacinusParacoccus marginatusmolecular identificationmolecular markerdroplet digital PCR
《昆虫学报》 2026 (2)
255-265,11
国家"十四五"重点研发计划项目(2021YFD1400100)海关总署科研项目(2024HK175,2023HK083)
评论