2种十字花科检疫性病菌快速检测方法的建立  

Establishment of rapid detection method for two quarantine pathogens in Cruciferae

作　　者：于璇 魏霜 姜子德[2] 王卫芳 YU Xuan;WEI Shuang;JIANG Zi-de;WANG Wei-fang(Guangzhou Customs Technology Center,Guangzhou 510623,China;College of Plant Protection,South China Agricultural University,Guangzhou 510640,China)

机构地区：[1]广州海关技术中心,广州510623 [2]华南农业大学植物保护学院,广州510640

出　　处：《西南农业学报》2023年第9期1962-1971,共10页Southwest China Journal of Agricultural Sciences

基　　金：海关总署科研项目(2020HK147);广州海关科研项目(2020GZCK⁃004)。

摘　　要：【目的】人工构建一段随机序列并作为扩增内标(Internal amplification control,IAC)添加进实时荧光PCR体系中,建立含有扩增内标的十字花科细菌性黑斑病菌(Pseudomonas syringae pv.maculicola)和油菜茎基溃疡病菌(Leptosphaeria maculans)的多重实时荧光PCR方法,检测上述2种检疫性病菌,旨在在提高检测通量的同时,指示PCR假阴性问题。【方法】通过对实时荧光PCR体系中各个引物及探针比例进行优化,从特异性、灵敏度以及实际样品检测等多个方面对该方法进行系统评价。【结果】在20.0μL体系中加入1.114×10^(3)拷贝的IAC不影响靶目标的扩增,特异性强,对十字花科细菌性黑斑病菌及油菜茎基溃疡病菌的灵敏度分别为6.9×10^(-4)、1.6×10^(-4)ng/μL。应用该方法对52批进口油菜籽和十字花科蔬菜种子样品进行检测,结果显示可以实现上述2种检疫性病菌的快速检测,同时有效指示PCR反应的假阴性。【结论】两种检疫性病菌含IAC多重实时荧光PCR检测方法不仅能提高检测准确性而且能防止漏检,在口岸进口种子检疫中具有良好的推广应用前景,对引种安全具有重要意义。【Objective】A random sequence was artificially constructed and added to real⁃time fluorescent PCR as internal amplification con⁃trol(IAC).A multiplex real⁃time PCR method containing Pseudomonas syringae pv.maculicola and Leptosphaeria maculans was estab⁃lished.The detection of the above two quarantine pathogens aimed to indicate the false negative problem of PCR and improve the detection throughput.【Method】By optimizing the proportion of primers and probes in real⁃time fluorescence PCR system,this method was systemati⁃cally evaluated from the aspects of specificity,sensitivity and actual sample detection.【Result】Adding 1.114×10^(3) copies of IAC into the 20μL system did not affect the amplification of the target,and had strong specificity.The sensitivity to P.syringae pv.maculicola and L.mac⁃ulans were 6.9×10^(-4) and 1.6×10^(-4) ng/μL,respectively.This method was used to detect 52 batches of imported rapeseed and cruciferous vegetable seed samples.The results showed that the method could realize the rapid detection of the above two kinds of quarantine pathogen and indicate the false negative of PCR reaction effectively at the same time.【Conclusion】This method can not only improve the accuracy of detection but also prevent missing detection.It has the prospect of popularization and application in the quarantine of imported seeds at ports,and is of great significance for the safety of seed introduction.

关 键 词：油菜籽 油菜茎基溃疡病菌 细菌性黑斑病菌 扩增内标 

分 类 号：S41[农业科学—植物保护]