| 摘要: |
| 以一株副溶血弧菌(Vibrio parahaemolyticus)作为研究对象，比较了MTT [3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]比色法、ATP生物发光法和高通量生长曲线法在活细菌高通量计数上的应用效果。用96孔培养板进行不同浓度细菌活菌计数，确定了上述3种方法在副溶血弧菌活菌计数的标准曲线和线性范围。结果显示，副溶血弧菌的MTT比色法以DMSO溶解的MTT产物甲瓒在555 nm的吸光度(OD555 nm)为计数依据，活菌数的对数(LgC)与LgOD555 nm线性关系的标准曲线为LgC=(1.0439±0.0200)LgOD555 nm+(8.0565±0.0125)，相关系数R²=0.9965，线性检测范围为7.8×106~2.5×108 CFU/ml；ATP生物发光法以ATP产生的相对发光度值(RLU)为计数依据，LgC与LgRUL线性关系的标准曲线为LgC=(0.9590±0.0065)LgRLU+(0.9949±0.0366)，相关系数R²=0.9994，线性检测范围为1.0×104~3.0×108 CFU/ml；高通量生长曲线法以生长曲线达到拐点的时间(Ts)为计数依据，LgC与Ts线性关系的标准曲线为LgC=(0.8727±0.0230)Ts+(9.0128±0.1572)，相关系数R²=0.9924，线性检测范围为1.0×100~1.0×107 CFU/ml。用3种方法对实际菌液测量并与平板计数法比较表明，ATP生物发光法与高通量生长曲线法有很好的准确性，MTT比色法准确度稍差，而高通量生长曲线法有最宽的线性范围，也最适合高通量测定。 |
| 关键词: 活菌计数 MTT比色法 ATP生物发光法 高通量生长曲线法 |
| DOI：10.19663/j.issn2095-9869.20180421001 |
| 分类号: |
| 基金项目:中国东盟海上合作基金项目(2016~2018)、现代农业产业技术体系(CARS-48)和中国水产科学研究院基本科研业务费专项(2017HY-ZD10)共同资助 |
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| Comparison of three high-throughput viable counting techniques for aquatic pathogenic bacteria |
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ZOU Peizhuo1,2,3,4,5,6, YANG Qian2,3,4,5,6, DONG Xuan2,3,4,5,6, XIE Guosi2,3,4,5,6, HUANG Jie1,2,3,4,5,6
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1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao);3.Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs;4.Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity;5.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;6.Qingdao 266071
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| Abstract: |
| Three viable counting methods, including MTT assay, ATP bioluminescence assay, and high-throughput growth curve assay, for live Vibrio parahaemolyticus were compared in a high-throughput manner on a 96-well culture plate. The standard curves and linear ranges of these three methods were determined. The results showed that the directly detected index of MTT assay is OD555 nm, which is the absorption at 555 nm of DMSO-dissolved formazan produced from MTT. The liner standard curve for viable V. parahaemolyticus between logarithmic viable counts (LgC) and the logarithmic OD555 nm (LgOD555 nm) was LgC=(1.0439±0.0200)LgOD555 nm+(8.0565±0.0125), with a correlation index R²= 0.9965, and the linear detection range is 7.8×106~2.5×108 CFU/ml. The directly detected index of ATP bioluminescence assay is the relative luminescence unit (RLU), which is produced by ATP of viable cells. The liner standard curve between LgC and LgRUL was LgC=(0.9590±0.0065)LgRLU+(0.9949±0.0366), with a correlation index R²=0.9994, and the linear detection range is 1.0×104~3.0×108 CFU/ml. The directly detected index of the high-throughput growth curve method is the spinodal time (Ts), which is the time reaching the reflection point on the growth curve. The liner standard curve between LgC and Ts was LgC=–(0.8727±0.0230)Ts+(9.0128±0.1572), with a correlation index R²=0.9924, and a linear detection range of 1.0×100~1.0×107 CFU/ml. These three methods were used to measure 10 broths of V. parahaemolyticus and comparison with the plate counting method, respectively. The results showed both the ATP bioluminescence assay and the high-throughput growth curve method had satisfying accuracy, but the MTT assay had lower accuracy. The high-throughput growth curve has the widest linear range and is the most suitable for high throughput measurement. |
| Key words: Viable counting MTT assay ATP bioluminescence assay High-throughput growth curve method |
