巢式PCR快速检测西瓜细菌性果斑病菌

doi:10.3864/j.issn.0578-1752.2014.02.008

摘要/Abstract

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5 µL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50 μL反应体系：5 μL 10×PCR buffer（25 mmol?L-1 MgCl2），4 μL dNTP （D4030RA，2.5 mmol?L-1），引物（5 μmol?L-1）各3 μL，0.4 μL 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3 μL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1 μL为模板，以引物BX-L1/BX-S-R2各3 μL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4.7×101 cfu/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。

关键词: 西瓜细菌性果斑病 , 西瓜种子 , PCR , 检测

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-L1/BX-R5 and BX-L1/BX-S-R2) derived from BOX short repeated sequence of Aac were selected to establish the nested-PCR. 5 µL 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 μL of 10× reaction buffer (25 mmol?L-1 MgCl2), 0.4 μL of Taq DNA polymerase (DR001B, 5 U?μL-1), 3 μL of each primer (5 μmol?L-1), 4 μL of dNTP（D4030RA, 2.5 mmol?L-1）, ddH2O was added to the final reaction volume of 50 μL. 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 μL 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-L1/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 carrier rate of infected seed was 0.1%-0.5% , the positive detection rate was 66.7%. When the carrier rate of infected seed was 1%-10% , the positive detection rate was 83%-100%.【Conclusion】The present study demonstrated that the procedure of nested-PCR is a sensitive, specific, rapid, reproducible method to detect Aac in watermelon seeds.

Key words: bacterial fruit blotch of watermelon , watermelon seed , PCR , detection

王婧, 毕阳, 朱艳, 韩舜愈, 祝霞, 盛文军, 李敏. 巢式PCR快速检测西瓜细菌性果斑病菌[J]. 中国农业科学, 2014, 47(2): 284-291.

WANG Jing, BI Yang, ZHU Yan, HAN Shun-Yu, ZHU Xia, SHENG Wen-Jun, LI Min. Nested-PCR Rapidly Detecte Acidovorax avenae subsp. citrulli from Watermelon Seeds[J]. Scientia Agricultura Sinica, 2014, 47(2): 284-291.

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