CN100432234C - 用含3-氨基苯酚硼酸的纸片检测细菌AmpC酶的方法 - Google Patents
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Abstract
本发明公开了属于生物细菌的检测技术范围的一种用含3-氨基苯酚硼酸的纸片检测细菌AmpC酶的方法。主要是利用硼酸能抑制AmpC酶的原理，先配制APB溶液，并在不加3-氨基苯酚硼酸(APB)的CAZ，CTX和FOX纸片上添加APB，采用APB纸片增强法进行纸片法药敏实验，检测细菌产生的AmpC酶。APB可使CAZ或/和CTX或/和FOX纸片对产生AmpC酶细菌的抑菌环扩大，因此通过测定对产生AmpC酶细菌抑菌环直径改变来判断AmpC酶的存在，避免使用单一底物可能带来的漏检，提高检测的阳性率。该方法具有操作简单，成本低廉，结果判读清晰，重复性好，不需要特殊实验条件的优点，易于在临床实验室推广应用。
Description
技术领域
本发明属于生物细菌的检测技术范围，特别涉及一种用含3-氨基苯酚硼酸(APB)的纸片检测细菌AmpC酶的方法。
背景技术
AmpCβ内酰胺酶，属于Ambler分类的C类酶(以下简称AmpC酶)是部分细菌如肺炎克雷伯菌、大肠埃希菌等在生长过程中产生的一种能水解三代头孢类抗生素(如头孢他啶CAZ、头孢噻肟CTX、头孢曲松CRO)、头霉素(如头孢西丁FOX)、单胺类抗生素(如氨曲南AZT)的酶。产生AmpC酶的细菌对以上抗生素表现耐药性，当这些细菌引起感染(如败血症、肺炎、肾盂肾炎或伤口感染)时，采用以上抗生素将会使治疗失败。因此，正确检测产生AmpC酶的细菌，对于了解细菌耐药机制、指导临床合理选用抗生素具有重要意义。但目前为止，尚缺乏简单、有效的方法供临床实验室用于对产生AmpC酶的细菌进行常规检测。三维试验检测AmpC酶(Coudron PE，Moland ES，Thomson KS.Occurrence and detectionof AmpC beta-lactamases among Escherichia coli，Klebsiella pneumoniae，and proteus mirabilis isolates at a veterans medical center.J ClinMicrobiol，2000，38：1791-1796)，需要制备酶的粗提物，并在琼脂平板上挖槽，操作烦琐，费时，且需要特殊设备如超声破碎仪或反复冻融条件。最新国外介绍的Tris-EDTA检测AmpC酶的方法(Black JA，Moland ES，and Thomson KS.AmpCdisk test for detection of plasmid-mediated AmpC β-lactamases inEnterobacteriaceae lacking chromosomal AmpC β-lactamases.J ClinMicrobiol，2005，43：3110-3113)虽然具有操作简单，成本低廉的特点，但需要制备含有Tris-EDTA及待检菌的纸片，增加操作步骤，且部分细菌的结果判读需要一定的经验。
发明内容
本发明的目的是提供一种用含3-氨基苯酚硼酸的纸片检测细菌AmpC酶的方法，其特征在于，该方法的具体检测步骤如下：
1)先配制APB溶液：用二甲基亚砜作为溶剂，配成浓度为40mg/ml的溶液；所述APB分子式为C6H8BNO2.H2O，分子量为15496；
2)取10μl上述APB溶液分别加入英国OXOID公司生产的每片含30μg CAZ、30μg CTX和30μg FOX的纸片上，使每片测试纸片上含APB 400μg，以制成CAZ/APB、CTX/APB和FOX/APB纸片；待纸片干后，置于2-8℃的温度下保存备用。
3)具体实验操作
(1)将新鲜培养的待检菌制成0.5麦氏单位菌悬液并均匀涂抹至M-H平板；
(2)将不含有APB的CAZ、CTX和FOX纸片及含有APB的CAZ/APB，CTX/APB和FOX/APB纸片分别贴于涂布有待检菌的M-H平板表面，35℃培养过夜，观察并分别记录含有APB的CAZ/APB，CTX/APB和FOX/APB纸片与不含APB的CAZ、CTX和FOX纸片抑菌环直径；
(3)结果判读：含有APB比不含APB的CAZ或/和CTX或/和FOX纸片抑菌环直径增加5mm或以上者判为AmpC酶阳性。
本发明的有益效果是该项检测是利用纸片扩散法药敏实验的特点，将AmpC酶抑制剂APB加至含有三种AmpC酶底物(CAZ，CTX，FOX)的药敏纸片上，通过测定加APB与不加APB的药敏纸片抑菌环直径改变来判断AmpC酶的存在，避免了使用单一底物可能带来的漏检，提高检测的阳性率。该实验还具有操作简单，成本低廉，结果判读清晰，重复性好，不需要特殊实验条件的优点，易于在临床实验室推广应用。
具体实施方式
本发明提供一种用含3-氨基苯酚硼酸的纸片检测细菌AmpC酶的方法。该方法的检测原理是由于硼酸是AmpC酶的抑制剂，本项发明在不加3-氨基苯酚硼酸APB的CAZ，CTX和FOX纸片基础上，添加APB至CAZ，CTX和FOX纸片，进行纸片法药敏实验，即APB纸片增强法，检测细菌产生的AmpC酶。APB可使CAZ或/和CTX或/和FOX纸片对产生AmpC酶细菌的抑菌环扩大，当含有APB的CAZ或/和CTX或/和FOX纸片比不含APB的CAZ或/和CTX或/和FOX纸片抑菌环直径增加5mm或以上时，判为AmpC酶检测阳性。
该方法的具体检测步骤如下：
1)先配制APB溶液：用二甲基亚砜作为溶剂，配成浓度为40mg/ml的溶液；所述APB分子式为C6H8BNO2.H2O，分子量为15496；
2)取10μl上述APB溶液分别加入英国OXOID公司生产的每片含30μg CAZ、30μg CTX和30μg FOX的纸片上，使每片测试纸片上含APB 400μg，以制成CAZ/APB、CTX/APB和FOX/APB纸片；待纸片干后，置于2-8℃的温度下保存备用。
3)具体实验操作
(1)将新鲜培养的待检菌制成0.5麦氏单位菌悬液并均匀涂抹至M-H平板；
(2)将不含有APB的CAZ、CTX和FOX纸片及含有APB的CAZ/APB，CTX/APB和FOX/APB纸片分别贴于涂布有待检菌的M-H平板表面，35℃培养过夜，观察并分别记录含有APB的CAZ/APB，CTX/APB和FOX/APB纸片与不含APB的CAZ、CTX和FOX纸片抑菌环直径；
(3)结果判读：含有APB比不含APB的CAZ或/和CTX或/和FOX纸片抑菌环直径增加5mm或以上者判为AmpC酶阳性。
举例说明
例1，男性，51岁，临床诊断为呼吸道感染，从其送检痰液标本中分离到肺炎克雷伯菌，用不含有APB的CAZ、CTX和FOX纸片及含有本发明制备的APB的CAZ/APB，CTX/APB和FOX/APB纸片分别贴于涂布有分离到肺炎克雷伯菌的M-H平板表面，35℃培养过夜，即采用纸片增强法检测结果如下：含有APB的CAZ/APB，CTX/APB和FOX/APB纸片的抑菌环直径分别是22mm，25mm和13mm；不含APB的CAZ、CTX和FOX纸片抑菌环直径分别是15mm，20mm和6mm。从而计算出含有APB比不含APB的CAZ，CTX和FOX纸片抑菌环直径分别增加了7mm，5mm和7mm，均在5mm以上。判定为AmpC酶阳性。故该病人是由产AmpC酶的肺炎克雷伯菌引起的呼吸道感染。
例2，女性，50岁，临床诊断为泌尿道感染，从其送检尿液标本中分离到大肠艾希菌，用与例1相同的两种纸片分别贴于涂布有分离到大肠艾希菌的M-H平板表面，35℃培养过夜，增强法检测，其结果如下：含有APB的CAZ/APB，CTX/APB和FOX/APB纸片的抑菌环直径分别是21mm，24mm和12mm；不含APB的CAZ、CTX和FOX纸片抑菌环直径分别是16mm，20mm和10mm。从而计算出含有APB比不含APB的CAZ，CTX和FOX纸片抑菌环直径分别增加了5mm，4mm和2mm，虽然仅CAZ/APB纸片比CAZ纸片抑菌环直径增加了5mm，仍然判定为AmpC酶阳性。故该病人是由产AmpC酶的大肠艾希菌引起的泌尿道感染。
Claims (1)
1.一种用含3-氨基苯酚硼酸的纸片检测细菌AmpC酶的方法，其特征在于，该方法的具体检测步骤如下：
1)先配制APB溶液：用二甲基亚砜作为溶剂，配成浓度为40mg/ml的溶液；所述APB分子式为C6H8BNO2.H2O，分子量为15496；
2)取10μl上述APB溶液分别加入英国OXOID公司生产的每片含30μg CAZ、30μg CTX和30μg FOX的纸片上，使每片测试纸片上含APB 400μg，以制成CAZ/APB、CTX/APB和FOX/APB纸片；待纸片干后，置于2-8℃的温度下保存备用；
3)具体实验操作
(1)将新鲜培养的待检菌制成0.5麦氏单位菌悬液并均匀涂抹至M-H平板；
(2)将不含有APB的CAZ、CTX和FOX纸片及含有APB的CAZ/APB，CTX/APB和FOX/APB纸片分别贴于涂布有待检菌的M-H平板表面，在35℃下培养过夜，观察并分别记录含有APB的CAZ/APB，CTX/APB和FOX/APB纸片与不含APB的CAZ、CTX和FOX纸片抑菌环直径；
(3)结果判读：含有APB比不含APB的CAZ或/和CTX或/和FOX纸片抑菌环直径增加5mm或以上者判为AmpC酶阳性。
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| Title |
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| AmpC酶和ESBLs介导的阴沟肠杆菌耐药. 佘丹阳.中国人民解放军军医进修学院博士学位论文. 2002 |
| AmpC酶和ESBLs介导的阴沟肠杆菌耐药. 佘丹阳.中国人民解放军军医进修学院博士学位论文. 2002 * |
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