生物学杂志

• 技术方法 • 上一篇    下一篇

利用MTT建立一种快速检测青枯菌活菌的方法

  

  1. 1. 重庆三峡学院 生命科学与工程学院, 万州 404120;2. 重庆三峡医药高等专科学校 基础医学部, 万州 404120
  • 出版日期:2019-02-18 发布日期:2019-02-18
  • 通讯作者: 熊书,讲师,主要从事分子生物学研究,E-mail:24594294@qq.com
  • 作者简介:周大祥,副教授,主要从事分子生物学研究,E-mail:dqzhou79@163.com
  • 基金资助:
    重庆市自然科学基金(cstc2016jcyjA0198)

Establishment of a method for rapid detecting only viable cells of Ralstonia solanacearum by MTT

  1. 1. College of Life Science & Engineering, Chongqing Three Gorges University, Wanzhou 404120; 2. Department of Basic Medicine, Chongqing Three Gorges Medical College, Wanzhou 404120, China
  • Online:2019-02-18 Published:2019-02-18

摘要: 利用四氮唑蓝(MTT),优化了检测波长、菌浓度、MTT用量、MTT反应时间、三联液的用量和溶解时间等,建立一种有效快速检测青枯菌活菌的方法。结果发现,MTT反应的最佳检测波长为512 nm,青枯菌浓度A600为0.3,MTT用量为30 μL,MTT还原反应控制在20 min以内,三联液溶解甲臜晶体的用量为3 mL,溶解8 h,甲臜溶液在10~120 μL以内与A512的数值线性关系良好,平板计数印证了这一结果。实验结果表明,MTT法可以用于快速检测青枯菌等植物病原菌活菌,对农产品进出口检验检疫具有一定的应用价值。

关键词: 青枯菌, MTT, 甲臜, 三联液, 活菌检测

Abstract: Method for assay of the viable cells of Ralstonia solanacearum was established by using methylthiazolyldiphenyl-tetrazolium bromide(MTT) and the parameters like the detection wavelength, bacterial concentration, MTT dosage and incubation time, triplex solution dosage and dissolution time were optimized. Results showed that the best detection wavelength for the reaction of MTT was 512 nm, Ralstonia solanacearum concentration was 0.3 in A600, the dosage of MTT was 30 μL, the reduction reaction of MTT was within 20 min, the dosage of triplex solution for dissolving formazan crystal was 3 mL, dissolved 8 h, and the A512 value was linear with formazan solutions between 10 μL and 120 μL. The study would provide the MTT method for rapid detection of plant pathogenic viable bacteria such as Ralstonia solanacearum, and have potential application value in agricultural products import and export detections.

Key words: Ralstonia solanacearum, MTT, formazan, triplex solution, detection of viable bacteria

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