AMB  >> Vol. 3 No. 2 (June 2014)

    酶标仪快速测定抗菌物质抑菌活性方法的建立
    Establishment of Rapid Determining Method for Antibacterial Activity by Microplate Reader

  • 全文下载: PDF(1343KB) HTML    PP.29-35   DOI: 10.12677/AMB.2014.32004  
  • 下载量: 2,577  浏览量: 8,923   科研立项经费支持

作者:  

周子雄:广东药学院中药学院,广州;
黄庆华,朱爽,周 林:广东药学院生命科学与生物制药学院,广东省生物技术候选药物研究重点实验室,广州

关键词:
酶标仪抑菌活性高通量筛选Microplate Reader Antimicrobial Activity High Throughput Screen

摘要:

对酶标仪法测定药物抑菌活性的主要影响因素进行研究,为基于酶标仪法快速筛选抑菌活性物质提供实验依据。以金黄色葡萄球菌标准菌株为受试菌株,测定代表性抗菌药物(盐酸卡那霉素、土霉素、氨苄青霉素、万古霉素),在不同培养方式(静置培养、振荡培养)、不同培养时间条件下,对金黄色葡萄球菌的抑菌率。结果表明药物浓度、培养方式、培养时间显著的影响抑菌活性的测定,在金黄色葡萄球菌浓度约5 × 105 cfu∙mL−1、37℃静置培养6 h的条件下,盐酸卡那霉素、土霉素、氨苄青霉素、万古霉素浓度为100 μg/mL时,抑菌率分别为57.4%、52.8%、57.4%、61.1%;浓度为10 μg/mL时,抑菌率分别为55.6%、36.1%、56.5%、56.0%;浓度为1 μg/mL时抑菌率分别为46.3%、10.0%、52.8%、50.0%。方法的精密度和重现性良好。在设定的测试条件下,酶标仪法与2010版中国药典中试管浊度法没有显著性差异,可用于抗菌活性物质的高通量筛选。

 To investigate the main factors influencing the antimicrobial activity by microplate reader method for high throughput screen antimicrobial substances, the inhibitory rate of staphylococcus aureus was determinated against four typical antibiotics (kanamycin hydrochloride, terramycin, ampicillin, vancomycin) under different culture conditions. The result showed that the concentration of the drug, culture conditions and culture time will affect the inhibitory rate of Staphylococcus aureus. With bacterial concentrations of 5 × 105 cfu∙mL−1, static culturing, incubation time of 6 h, the inhibitory rate of kanamycin hydrochloride, terramycin, ampicillin, vancomycin was 57.4%, 52.8%, 57.4%, 52.8% respectively with the concentration of 100 μg/mL antibiotic, while the inhibitory rate was 55.6%, 36.1%, 56.5%, 36.1% respectively with the concentration of 10 μg/mL antibiotic, and the inhibitory rate was 46.3%, 10.0%, 52.8%, 50.0% respectively with the concentration of 1 μg/mL antibiotic. The precision and repeatability of the assay were good. There is no significant difference, in the inhibitory rate, between the microplate reader method and tube turbidimetry method from 2010 China Pharmacopoeia. The established microplate reader method could be applied to high throughput screen of antibacterial substances.

文章引用:
周子雄, 黄庆华, 朱爽, 周林. 酶标仪快速测定抗菌物质抑菌活性方法的建立[J]. 微生物前沿, 2014, 3(2): 29-35. http://dx.doi.org/10.12677/AMB.2014.32004

参考文献

[1] 高燕霞, 姜建国, 王茉莉 (2009) 浊度法测定土霉素原料药及其片剂的效价. 中国药房, 20, 290-291.
[2] 肖婷, 刘守柱, 薛超彬, 等 (2008) 紫外分光光度计法与酶标仪微量法测定酚氧化酶蛋白含量及活力的比较. 昆虫知识, 45, 306-309.
[3] 薛超彬, 陈清西, 王勤, 等 (2004) 菜青虫不同虫态及虫龄的多酚氧化酶性质比较. 昆虫学报, 47, 305-309.
[4] Kohji, O., Yuhsuke, W., Tetsuo, U., et al. (2003) Catalyst development for methanol synthesis using parallel react ors for high-through put screening based on a 96 well micro plate system. Journal of the Japan Petroleum Institute, 46, 328-334.
[5] 王树栋, 罗万春, 高兴祥, 等 (2004) 曲酸对小菜蛾酚氧化酶抑制作用的研究. 中国农业科学, 37, 1316-1321
[6] 余久如, 黎增文, 潘桂红, 等 (2011) 酶标仪自检方法的建立和应用评价. 现代检验医学, 31, 112-114.
[7] 范鹏, 蒋林东, 王小丹, 等 (2010) 紫外分光光度计与酶标仪测定盐酸川芎嗪含量的比较. 西南国防医药, 23, 223-229.
[8] 骆健美, 成永新, 李培君, 等 (2011) 基于抑菌活性的ε-聚赖氨酸的微孔板生物检测法. 微生物学通报, 38, 976-981.
[9] 陈默, 王志伟, 胡长鹰, 等 (2009) 酶标仪法快速评价香兰素的抑菌活性. 食品与发酵工业, 35, 63-66.
[10] 国家药典委员会 (2010) 中华人民共和国药典2010版. 中国医药科技出版社, 北京, 附录95.
[11] 杨志俊 (2004) 酶标仪单、双波长比色测定OD值在卫检实验中的应用探讨. 口岸卫生控制, 9, 5-6.
[12] 杨帆, 李淑梅, 杨晓, 等 (2011) 分光光度法与酶标仪微量法测量菌液浓度的比较. 光谱实验室, 28, 1752-1754.