酶标仪快速测定抗菌物质抑菌活性方法的建立

Abstract

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.