巢式PCR快速检测西瓜细菌性果斑病菌  

Nested-PCR Rapidly Detecte Acidovorax avenae subsp. citrulli from Watermelon Seeds

作　　者：王婧[1] 毕阳[1] 朱艳[1] 韩舜愈[1] 祝霞[1] 盛文军[1] 李敏[1] 

机构地区：[1]甘肃农业大学食品科学与工程学院,兰州730070

出　　处：《中国农业科学》2014年第2期284-291,共8页Scientia Agricultura Sinica

基　　金：国家质检总局项目(2009IK276)

摘　　要：【目的】建立nested-PCR方法,快速检测西瓜种子中的燕麦嗜酸菌西瓜亚种(Acidovorax avenae subsp.citrulli,Aac),为西瓜细菌性果斑病(bacterial fruit blotch of watermelon,WFB)的防控提供技术支持。【方法】根据Aac BOX短重复序列的PCR产物设计两对引物BX-L1/BX-R5和BX-L1/BX-S-R2,建立以BX-L1/BX-R5为外侧引物,BX-L1/BX-S-R2为内侧引物的nested-PCR,以5μL样品处理液于99℃高温裂解10 min,再放置冰上冷却5 min,以所释放病原DNA为模板进行PCR扩增,采用50μL反应体系:5μL 10×PCR buffer(25 mmol·L-1MgCl2),4μL dNTP(D4030RA,2.5 mmol·L-1),引物(5μmol·L-1)各3μL,0.4μL Taq DNA酶(DR001B,5 U·μL-1),通过退火温度优化,反应条件为:引物BX-L1/BX-R5各3μL进行第一轮扩增,95℃,2 min;95℃,30 s;65℃,45s;72℃,1 min;35个循环;72℃,延伸7 min;取扩增后的产物1μL为模板,以引物BX-L1/BX-S-R2各3μL进行第二轮扩增,95℃,2 min;95℃,30 s,66℃,45 s,72℃,1 min,30个循环,72℃,7 min。在此条件下对梯度Aac菌悬液、模拟带菌种子提取液进行特异性、灵敏性及重复性检验,对不同带菌率的西瓜种子提取液进行检测。【结果】引物BX-L1/BX-R5和BX-L1/BX-S-R2在检测不同来源的Aac菌株时都产生了预期大小的片段,并且对其近源种燕麦嗜酸菌卡特莱兰亚种(A.avenae subsp.cattleyae)、魔芋假单胞菌(A.avenae subsp.konjaci)及其不相关菌株未扩增出目标片段。以BX-L1/BX-R5为外侧引物,BX-L1/BX-S-R2为内侧引物的nested-PCR,对Aac纯菌液和模拟带菌种子提取液的最低检测限4.7×101cfu/mL,比direct-PCR灵敏度高出1 000倍。当西瓜种子带菌率在0.1%—0.5%时,nested-PCR阳性检测率为66.7%;当种子带菌率为1%—10%时,nested-PCR的阳性检测率为83%—100%。【结论】以BX-L1/BX-R5为外侧引物,BX-L1/BX-S-R2为内侧引物的nested-PCR方法,能够快速、高效检测携带微量Aac的西瓜种子,检测结果重现性高。Abstract： [Objective] The objective of this study is to develop a nested-PCR method to rapidly and accurately detect Aac （Acidovorax avenae subsp, citrulli） in watermelon seeds, and to provide technique support for prevention and control bacterial fruit blotch of watermelon （WFB）. [ Method ] Two pairs of specific primer sets （BX-L 1/BX-R5 and BX-L 1/BX-S-R2） derived from BOX short repeated sequence of Aac were selected to establish the nested-PCR. 5 μLL of suspension were added in the tubes, boiled at 99℃for 10 min, and then chilled on ice for 5 min, the released pathogen DNA was used as template for PCR reaction. PCR reaction system was performed with 5 gL of 10× reaction buffer （25 mmol·L-I MgC12）, 0.4 μL ofTaq DNA polymerase （DR001B, 5 μL Ll）, 3 μL of each primer （5 grnol.Ll）, 4 gL of dNTP （D4030RA, 2.5 mrnol·L-1 ） , ddH20 was added to the final reaction volume of 50 gL. DNA amplification was performed with 2 min at 95 ℃, followed by 35 cycles consisting of denaturation （30 s at 95℃）, annealing （45 s at 65℃） extension （1 min at 72℃）, and a final extension at 72℃ for 7 min. The nested-PCR was performed using （BX-L1/BX-R5 primers for the first run and the BX-L1/BX-S-R2 sets for the second run by using 1 ktL of the first PCR product as the template applying the same thermal profile and number of cycles and annealing （45 s at 66℃）. Analytical sensitivity, specificity and reproducibility were assessed, respectively. The nested-PCR method was used to detect a series of dilution of Aac suspension and different bacteria-carrying rates of seeds. [Result] Aac in different strains was amplified by the specific primer sets BX-L1/BX-R5 and BX-LI/BX-S-R2, respectively. A target band was amplified from Aac strains but not from A. avenae subsp, cattleyae, A. avenae subsp, konjaci and other bacteria. The nested-PCR assay had a lowest detection limit of 4.7× 101 cfu/mL, the sensitivity was 1 000 times higher than the conventional direct-PCR. When the carder r

关 键 词：西瓜细菌性果斑病 西瓜种子 PCR 检测 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]