Microtox中药注射剂微毒测试体系明亮发光杆菌502和青海弧菌Q67发光反应条件的比较研究

Reaction conditions comparison of Photosbacterium phosphoreum 502 and Vibrio qinghaiensis Q67 in Microtox-based fast testing system for traditional Chinese medicine injection

目的:基于明亮发光杆菌502和青海弧菌Q67的Microtox微毒测试体系比较研究,明确两者适宜的反应条件。方法:1考察明亮发光杆菌502和青海弧菌Q67在15℃±1℃、20℃±1℃、25℃±1℃下的相对发光情况;2采用Plackett-Burman法优化设计影响明亮发光杆菌502和青海弧菌Q67发光的因素;3采用Plackett-Burman法优化得到的反应体系,考察不同p H复苏液对上述两种细菌相对发光强度的影响。结果:1明亮发光杆菌502的相对发光强度随温度的升高而增强,而青海弧菌Q67的相对发光强度随温度的升高先增强后减弱。2Plackett-Burman法得到最优发光条件为:明亮发光杆菌502冻干粉平衡10min后加入复苏液2ml,混匀15min,取100μl菌液测试初始发光度,随后立即加入1ml复苏液或待测样品,反应15min后再次测试相应发光强度;青海弧菌Q67冻干粉平衡10min后加入复苏液2ml,混匀10min,取100μl菌液测试初始发光度,随后立即加入2ml复苏液或待测样品,反应10min后再次测试相应发光强度。3复苏液p H 3.6-p H 3.8时对明亮发光杆菌502发光的影响由抑制转为增强;复苏液p H 4.5-p H 5.0时对青海弧菌Q67发光强度的影响<±15%。结论:与明亮发光杆菌502比较,青海弧菌Q67具有更宽的p H耐受范围并且在检测时无需调节待测样品渗透压。

Objective： Microtox assay system based on Photosbacterium phosphoreum 502（ P. p. 502） and Vibrio qinghaiensis Q67（ V. q. Q67）were compared to definite suitable reaction conditions. Method： 1 The relative luminous intensity of P. p. 502 and V. q. Q67 were investigated under 15℃ ± 1℃,20℃ ± 1℃ and 25℃ ± 1℃. 2The Plackett- Burman trials were carried out to optimize the agents which affected P. p. 502 and V. q. Q67. 3 The p H ranges suitable for each assay system was also investigated. Results： 1 With increased temperature,relative luminous intensity enhanced in P. p. 502 while enhanced first and then fell in V. q. Q67. 2 At a temperature of 15℃ ± 1℃,P. p.502 vials were placed at a thermostat for 10 min,then,2ml rehydration solution（ 3% Na Cl solution） was pour quickly into each vial and blended them into suspensions. After a waiting time of 15 min,added 100 μl bacteria suspension into the test tube to measure the luminescence intensity,immediately before the addition of 1 ml rehydration solution or test samples. The same time intervals（ 10s） were used for later intensity measurement. After a contact time of 15 min,the luminescence intensity of each vial was measured again. At a temperature of15℃ ± 1℃,V. q. Q67 vials were placed at a thermostat for 10 min,then,2 ml rehydration solution（ 0. 85% Na Cl solution） was pour quickly into each vial and blended them into suspensions. After a waiting time of 10 min,added 100 μl bacteria suspension into the test tube to measure the luminescence intensity,immediately before the addition of 2 ml rehydration solution or test samples. The same time intervals（ 10s） were used for later intensity measurement. After a contact time of 10 min,the luminescence intensity of each vial was measured again.3 p H 3. 6- p H 3. 8 reversed luminescence intensity of P. p. 502 from inhibition to enhancement. p H 4. 5- p H 5. 0 inhibited lower than ±15% luminescence intensity of V. q. Q67. The suitable reaction conditions of the two Microtox assay systems based on P. p. 502 and V. q.Q67 were absolutely different from each other. Moreover,V. q. Q67 is superior to P. p. 502 in p H tolerance and osmolarity adjustment needless.