三维法检测β内酰胺酶的影响因素探讨及方法的改进

An investigation of factors influencing three dimensional test for be ta-lactamases

目的 探讨影响三维法检测 β内酰胺酶结果的各种因素。方法 采用琼脂和肉汤两种不同增菌法及冻融和超声两种不同裂解菌体方法制备酶粗提液 ,以头孢他啶、头孢噻肟、头孢曲松、头孢泊肟、头孢吡肟、氨曲南、亚胺培南和头孢西丁为底物 ,克拉维酸、邻氯西林、EDTA为酶抑制剂 ,用三维试验法检测 β内酰胺酶。 结果 琼脂增菌与肉汤增菌的结果一致 ,-10～ -18℃ ,冻融5～ 9次与超声法对菌体的裂解效果无差别。菌量 <10 11CFU/ml ,冻溶液为生理盐水、磷酸盐缓冲液、无菌蒸馏水 ,冻融 1～ 3次残余菌量 >10 6 CFU/ml,水解底物、沟槽与抗生素纸片间的距离 >10mm ,抑制剂浓度 <10mmol/L ,加样量 <2 0 μl时 ,对试验结果的影响较大。菌龄 2 4～ 96h、冻融温度 -10～ -80℃、抑制剂与粗提酶液作用时间 0～ 90min、贴抗生素纸片与加酶液时间 5～ 90min、粗提酶液保存条件 4℃、-2 0℃、-3 5℃、-80℃等因素对试验结果影响不大。结论 三维试验法是检测质粒介导

Objective To investigate factors influencing the three dimensional test for beta lactama ses. Methods The bacteria were cultured in agar and broth media and lysed by freeze thawing -10℃～-80℃, 5～9 cycle and sonicating f or the preparation of the lactamase solutions. The enzyme activity was tested by the three dimensional method under various conditions. The substrates used wer e ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, cefepime, aztreonam, imipen em,and cefoxitin. The inhibitors used were clavulanic acid, cloxacillin and EDT A. Results No significant difference was found in results obtained with different culture media and different lysis procedures. Major fac tors influencing the test results were bacteria quantity <10 11 CFU/ml, fre ezing solution type NS?PBS?WS, the number of freeze thawing 1～3 cycle, the q uantity of the residue bacteria >10 6 CFU/ml, substrate type, the distance betw een sample and substrate >10 mm, inhibitor concentration <1 0 mmol/L and the am ount of sample loaded <20 μl. The test procedure was not sensitive to bacteria age 24～96 h, temperatures for freezing and thawing -10℃～-80℃, reaction time of the inhibitor with the enzyme 0～90 min, time lag between sample and substrat e loadings 5～90 min and storage conditions of the enzyme solutions 4℃?-20℃? -35℃?-80℃. Conclusion The three dimensional test is a simple and reliable method for detecting plasmid mediated beta lactamases.