| 摘要: |
| 【目的】建立苹果黑星病菌（ Venturia inaequalis(Cooke)Wint）的分子检测方法，以提高检测精确度，缩短检测时间。【方法】根据苹果黑星病菌与其他苹果病原真菌核糖体基因转录间隔区（Internal transcribed spacer, ITS）序列间的差异，设计了1对特异性引物320A/320B，用于苹果黑星病菌的分子检测，对特异性引物扩增条件进行了优化，并验证和检验了引物的特异性和灵敏度。【结果】特异性引物的扩增条带约为320 bp，优化的反应体系为：2 μL MgCl2(25 mmol/L)，2.5 μL 10×buffer，3 μL dNTP(2.5 mmol/L)，1.2 μL 引物320A/320B (10 μmol/L)，0.5 μL 聚合酶(5 U/μL)，1 μL模版DNA（30 ng/μL），加ddH2O至总体积25 μL；反应程序为：94 ℃ 3 min；94 ℃变性30 s，60 ℃退火30 s，72 ℃延伸30 s，35个循环；72 ℃延伸10 min。利用该对特异性引物对包括苹果黑星病菌在内的26个苹果病原菌菌株基因组DNA进行的PCR扩增表明，只有苹果黑星病菌能扩增到1条约320 bp的特异性条带，其他菌株及阴性对照的扩增产物均未检测到特异性条带。对接种苹果黑星病菌的苹果组织的检测表明，该对引物能特异性地检测到苹果黑星病菌的存在，其对苹果黑星病菌基因组DNA检测的灵敏度为100 fp/μL。【结论】利用设计的特异性引物320A/320B，参考正交试验优化的体系和程序，结合简单的SDS法提取苹果黑星病菌基因组DNA,在1个工作日内即可完成对该病原菌的分子检测。 |
| 关键词: 苹果黑星病菌 转录间隔区 特异性引物 正交优化 |
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| 基金项目:教育部长江学者和创新团队发展计划项目（200558）；欧盟科技合作项目（ICA4-CT-2001-10001）；教育部“111”引智项目（B07049） |
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| Molecular detection of Venturia inaequalis |
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| Abstract: |
| 【Objective】The molecular detection method was established to improve accuracy and shorten time of detection.【Method】According to the difference of internal transcribed spacer (ITS) between Venturia inaequalis (Cooke)Wint and fungi of other diseases for apple,a specific primer set (320A/320B) was designed for molecular detection of V.inaequalis.The genomic DNA of 26 fungal strains,including V.inaequalis,were amplified by PCR using this primer set.The result showed that the primer set that allowed the amplification of a 320 bp DNA fragment within the ITS region was specific to V.inaequalis.【Result】The specific primer set is about 320 bp,and its PCR-reaction system optimized was performed with a total volume of 25 μL reaction mixture containing 2 μL MgCl2(25 mmol/L),2.5 μL 10×buffer,3 μL dNTP(2.5 mmol/L),1.2 μL forward and reverse primer 320A/320B (10 μmol/L),0.5 μL Taq polymerase (5 U/μL),1 μLDNA template(30 ng/μL),and ddH2O.Besides,PCR was carried out in a thermocycler programmed for 35 cycles consisting of a denaturing step at 94 ℃ for 30 s,and annealing step at 60 ℃ for 30 s,and an extension step at 72 ℃ for 30 s.These cycles were preceded by an initial denaturation step at 94 ℃ for 3 min,and ended with a final extension at 72 ℃ for 10 min.Then,for detection of inoculated apple tissue,the result indicated that the genomic DNA of V.inaequaliscan be checked.The sensitivity of the primer set for genomic DNA of V.inaequalis was 100 fp/μL.【Conclusion】Using the 320A/320B PCR-reaction system,programme of the primer set for V.inaequalis was optimized by orthogonal design optimization,and adopting the measure of SDS for extracting the DNA,the molecular identification of inoculated plant tissue can be finished in only one working day. |
| Key words: Venturia inaequalis ITS primer set optimization |
