基于PCR和巢式PCR技术的玉米南方锈病早期检测  

Early Molecular Diagnosis of Southern Corn Rust Based on Conventional PCR and Nested PCR Assays

作　　者：马红霞[1] 孙华[1] 郭宁[1] 刘树森 张海剑[1] 石洁[1] MA HongXia;SUN Hua;GUO Ning;LIU ShuSen;ZHANG HaiJian;SHI Jie(Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences/Key Laboratory of IPM on Crops in Northern Region of North China,Ministry of Agriculture and Rural Affairs/IPM Centre of Hebei Province,Baoding 071000,Hebei)

机构地区：[1]河北省农林科学院植物保护研究所/农业农村部华北北部作物有害生物综合治理重点实验室/河北省农业有害生物综合防治工程技术研究中心,河北保定071000

出　　处：《中国农业科学》2023年第9期1686-1695,共10页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系(CARS-02);河北省重点研发计划(20326501D)。

摘　　要：【目的】建立巢式PCR方法,快速检测处于病害潜育期的玉米叶片内的多堆柄锈菌(Puccinia polysora),为玉米南方锈病(southern corn rust)的预测和防治提供技术支持。【方法】根据多堆柄锈菌ITS区序列,在其变异区设计3条多堆柄锈菌特异性检测引物NX471-F、NX255-F和NX255-R,建立以NX471-F与真菌ITS区通用引物ITS4为外侧引物,NX255-F/NX255-R为内侧引物的巢式PCR。采用20μL扩增体系:Ex Taq DNA聚合酶(5 U·μL~(-1))0.15μL、10×Ex Taq Buffer(Mg~(2+)plus)2μL,d NTP Mixture(各2.5 mmol·L~(-1))1.6μL,上下游引物(10μmol·L~(-1))各0.3μL,DNA 1μL,14.65μL ddH_2O。扩增程序:利用外侧引物NX471-F/ITS4进行第一步PCR扩增,95℃预变性7 min;95℃变性30 s,55℃退火30 s,72℃延伸45 s,30个循环;72℃再延伸7 min。将扩增后的产物稀释20倍,取1μL用作第二步PCR的扩增模板,然后以内侧引物NX255-F/NX255-R进行第二步PCR扩增,95℃预变性7 min;95℃变性30 s,66℃退火30 s,72℃延伸26 s,30个循环;72℃再延伸7 min。同时选用内侧引物进行常规PCR扩增,扩增体系同巢式PCR,扩增程序除循环数为38,其余程序同巢式PCR的第二步PCR检测。在此条件下对多堆柄锈菌、高粱柄锈菌(Puccinia sorghi)、青杨叶锈病菌(Melampsora laricipopulina)和7种玉米常见病原菌进行特异性检测,对不同梯度多堆柄锈菌基因组DNA和3种人工接种病叶进行灵敏度检测。【结果】巢式PCR检测体系仅在检测多堆柄锈菌时,产生了255 bp的目的条带,其他菌株均未扩增出目的条带。巢式PCR检测体系对多堆柄锈基因组DNA的最低检测限为10 fg·μL~(-1),是常规PCR检测体系灵敏度的500倍。当1 g人工接种病叶含500—2.5×10~4个多堆柄锈菌夏孢子时,常规PCR和巢式PCR检出阳性率分别0和85.71%,巢式PCR可检测出的夏孢子个数最少为1 000个;当1 g人工接种病叶中含1—7个夏孢子堆时,常规PCR和巢式PCR检出阳性率分别76.19%和100%,常规PCR可检�【Objective】The objective of this study is to establish a rapid detection method for Puccinia polysora during the incubation period based on the nested PCR,and to provide support for prediction and control of southern corn rust.【Method】The nested PCR primers for the specific detection of P.polysora were designed using the variant region of the ITS sequence,including outer primer NX471-F/ITS4 and inner primer NX255-F/NX255-R.For amplification,the 20µL PCR reaction mixture contained:Ex Taq DNA polymerase(5 U·μL-1)0.15μL,10×Ex Taq Buffer(Mg2+plus)2μL,dNTP Mixture(2.5 mmol·L-1 each)1.6μL,forward and reverse primers(10μmol·L-1)0.3μL each,template DNA 1μL,ddH2O 14.65μL.The PCR program was performed as follows:the outer primer NX471-F/ITS4 was used for the first amplification and denaturation at 95℃for 7 min,30 cycles of denaturation at 95℃for 30 s,annealing at 55℃for 30 s,extension at 72℃for 45 s,and a final extension at 72℃for 7 min.The product was diluted 20 times and used as the template for the second run of the nested PCR,which was amplified with the inner primer NX255-F/NX255-R,and the PCR program was denaturation at 95℃for 7 min;30 cycles of 95℃for 30 s,66℃for 30 s,and 72℃for 26 s;72℃for 7 min.At the same time,the inner primer was selected for conventional PCR,in which the reaction mixture was the same as the nested PCR,and the amplification conditions were the same as the second run,except that the number of reaction cycles was 38.Under these conditions,the specificity was detected for P.polysora,Puccinia sorghi,Melampsora laricipopulina and seven other common maize pathogens,and the sensitivity of nested PCR was tested using genomic DNA of P.polysora and DNA from artificially inoculated leaves.【Result】Nested PCR could specifically detect P.polysora from all tested fungi,with a 255 bp target fragment.The lowest detection limit of the nested PCR was 10 fg·μL-1,and the sensitivity was 500 times that of the conventional PCR.For the samples containing 500-2.5×104 uredi

关 键 词：玉米南方锈病 多堆柄锈菌 常规PCR 巢式PCR 

分 类 号：S435.131[农业科学—农业昆虫与害虫防治]