CN110438214A - Cre菌株碳青霉烯酶检测方法的性能评估方法 - Google Patents
Cre菌株碳青霉烯酶检测方法的性能评估方法 Download PDFInfo
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Abstract
CRE菌株碳青霉烯酶检测方法的性能评估方法,包括以下步骤;步骤1:检测碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;步骤2:改良Hodge试验以厄他培南为检测底物,产碳青霉烯酶;步骤3:取TSB肉汤,一管为mCIM试验管,另一管加入EDTA溶液作为eCIM试验管,用接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述TSB肉汤中,震荡混匀;得到得到含有受试菌的菌液;步骤4:将美罗培南纸片浸入菌液中;步骤5:制备菌悬液并均匀涂布M‑H琼脂平板,干燥;步骤6:接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M‑H琼脂板上,温育,量取抑菌圈直径;本发明提高对耐碳青霉烯酶菌株筛选的有效性。
Description
技术领域
本发明涉及碳青霉烯酶检测技术领域,特别涉及CRE菌株碳青霉烯酶检测方法的性能评估方法。
背景技术
现有的评估方法主要是改良Hodge试验。改良Hodge试验是一个表型筛选实验,不能对A、B、D类酶进行区分
现有的耐碳青霉烯酶分为A、B、D类酶,每种酶耐药机制及水平不同,改良Hodge试验检测不了。且改良Hodge试验的阳性率较高,有假阳性菌株的检出。
发明内容
为了解决以上技术问题,本发明的目的在于提供CRE菌株碳青霉烯酶检测方法的性能评估方法,mCIM(改良碳青霉烯酶类失活法)及eCIM(EDTA碳青霉烯酶类失活法)协同检测提高对耐碳青霉烯酶菌株筛选的有效性。
为了实现上述目的,本发明采用的技术方案是:
CRE菌株碳青霉烯酶检测方法的性能评估方法,包括以下步骤;
步骤1:VITEK2鉴定的亚胺培南耐药菌株复苏后用加热煮沸法提取DNA模板,检测5个碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;
步骤2:改良Hodge试验以厄他培南为检测底物,检测菌株划线与大肠埃希菌抑菌环交叉处细菌有增强生长的趋势判为阳性,即产碳青霉烯酶;
步骤3:mCIM和eCIM:取2管2mL TSB肉汤,一管为mCIM试验管,另一管加入20μL0.5mol/L EDTA溶液作为eCIM试验管,用1μL接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述2管TSB肉汤中(mCIM管和eCIM管),震荡混匀;得到得到2管含有受试菌的菌液;
步骤4:将美罗培南纸片(10μg)浸入菌液中;
步骤5:制备0.5麦氏浊度单位大肠埃希菌ATCC 25922菌悬液并均匀涂布M-H琼脂平板,干燥;
步骤6:用10μL接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M-H琼脂板上,同一受试菌mCIM和eCIM试验管中的美罗培南纸片贴于同一M-H平板,温育,量取抑菌圈直径;
步骤7:mCIM结果判断:
碳青霉烯酶阳性:美罗培南抑菌圈直径为6-15mm或直径为16-18mm但抑菌圈内有散在菌落;碳青霉烯酶阴性:抑菌圈直径≥19mm;碳青霉烯酶中性:抑菌圈直径为16-18mm,或直径为≥19mm但抑菌圈内有散在菌落。
eCIM结果判断:金属酶阳性:与mCIM结果相比,美罗培南抑菌圈直径≥5mm(如mCIM=6mm,eCIM=15mm,抑菌圈直径之差为9mm);金属酶阴性:与mCIM结果相比,美罗培南抑菌圈直径≤4mm(如mCIM=6mm,eCIM=8mm,抑菌圈直径之差为2mm)。
所述的EDTA溶液终浓度为5mmol/L。
所述的步骤4中将美罗培南纸片浸入菌液中,35℃温育4h。
所述的步骤5中干燥3~10min。
所述的步骤6中35℃温育18~24h。
本发明的有益效果:
改良Hodge试验对耐药菌株的筛选存在假阳性结果。与改良Hodge试验相比较,mCIM对验室人员操作和技术因素等影响较小,成本低廉,结果易于判读。mCIM试验是检测肠杆菌科细菌产生碳青霉烯酶一种简单、廉价、准确可靠的方法;与eCIM联同实验能较为准确的对A类酶和B类酶进行鉴别。
附图说明
图1为部分菌株改良Hodge试验结果示意图。
图2为部分菌株mCIM结果示意图。
图3为部分菌株eCIM结果示意图。
图4为丝氨酸碳青霉烯酶阳性结果示意图。
图5为金属酶阳性结果示意图。
图6为碳青霉烯酶阴性结果示意图。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
实施例1:
步骤1:VITEK2鉴定的亚胺培南耐药菌株复苏后用加热煮沸法提取DNA模板,检测5个碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;
步骤2:改良Hodge试验以厄他培南为检测底物,检测菌株划线与大肠埃希菌抑菌环交叉处细菌有增强生长的趋势判为阳性,即产碳青霉烯酶;
步骤3:mCIM和eCIM:取2管2mL TSB肉汤,一管为mCIM试验管,另一管加入20μL0.5mol/L EDTA溶液(终浓度为5mmol/L)作为eCIM试验管,用1μL接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述2管TSB肉汤中(mCIM管和eCIM管),震荡混匀;得到得到2管含有受试菌的菌液;
步骤4:将美罗培南纸片(10μg)浸入菌液中,35℃温育4h;
步骤5:制备0.5麦氏浊度单位大肠埃希菌ATCC 25922菌悬液并均匀涂布M-H琼脂平板,干燥3min;
步骤6:用10μL接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M-H琼脂板上,同一受试菌mCIM和eCIM试验管中的美罗培南纸片贴于同一M-H平板,35℃温育18h,量取抑菌圈直径。
实施例2:
步骤1:VITEK2鉴定的亚胺培南耐药菌株复苏后用加热煮沸法提取DNA模板,检测5个碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;
步骤2:改良Hodge试验以厄他培南为检测底物,检测菌株划线与大肠埃希菌抑菌环交叉处细菌有增强生长的趋势判为阳性,即产碳青霉烯酶;
步骤3:mCIM和eCIM:取2管2mL TSB肉汤,一管为mCIM试验管,另一管加入20μL0.5mol/L EDTA溶液(终浓度为5mmol/L)作为eCIM试验管,用1μL接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述2管TSB肉汤中(mCIM管和eCIM管),震荡混匀;得到得到2管含有受试菌的菌液;
步骤4:将美罗培南纸片(10μg)浸入菌液中,35℃温育4h;
步骤5:制备0.5麦氏浊度单位大肠埃希菌ATCC 25922菌悬液并均匀涂布M-H琼脂平板,干燥10min;
步骤6:用10μL接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M-H琼脂板上,同一受试菌mCIM和eCIM试验管中的美罗培南纸片贴于同一M-H平板,35℃温育24h,量取抑菌圈直径。
实施例3:
步骤1:VITEK2鉴定的亚胺培南耐药菌株复苏后用加热煮沸法提取DNA模板,检测5个碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;
步骤2:改良Hodge试验以厄他培南为检测底物,检测菌株划线与大肠埃希菌抑菌环交叉处细菌有增强生长的趋势判为阳性,即产碳青霉烯酶;
步骤3:mCIM和eCIM:取2管2mL TSB肉汤,一管为mCIM试验管,另一管加入20μL0.5mol/L EDTA溶液(终浓度为5mmol/L)作为eCIM试验管,用1μL接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述2管TSB肉汤中(mCIM管和eCIM管),震荡混匀;得到得到2管含有受试菌的菌液;
步骤4:将美罗培南纸片(10μg)浸入菌液中,35℃温育4h;
步骤5:制备0.5麦氏浊度单位大肠埃希菌ATCC 25922菌悬液并均匀涂布M-H琼脂平板,干燥7min;
步骤6:用10μL接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M-H琼脂板上,同一受试菌mCIM和eCIM试验管中的美罗培南纸片贴于同一M-H平板,35℃温育20h,量取抑菌圈直径。
1.收集的菌株信息筛选出47株CRE菌株,同时收集15株对碳青霉烯类抗生素敏感的肠杆菌科细菌做对照组。
2.PCR结果显示47株CRE菌株均为阳性,其中产KPC有44株,NDM有5株,IMP有1株;同时含有KPC和NDM两种基因型的菌株有3株,未检测到VIM和OXA48基因型;对照组细菌PCR全部阴性。
3. 47株CRE菌株改良Hodge试验全为阳性,对照组细菌14株阴性,1株阳性;敏感性为100%,特异性为93.33%;47株CRE菌株mCIM试验全部阳性,对照组细菌均为阴性;敏感性与特异性为100%;PCR检测47株CRE中有6株含有B类碳青霉烯酶基因,与mCIM实验联同进行的eCIM检测6株均为阳性,对照组细菌均为阴性;与PCR结果相一致,敏感性与特异性为100%。
利用VITEK 2 Compact全自动微生物鉴定系统挑选出对厄他培南、亚胺培南和美罗培南耐药的肠杆菌科细菌,并用纸片扩散法(KB法)进行药敏确认试验。利用PCR检测结果为金标准,评估改良Hodge试验、mCIM及eCIM在筛选CRE菌株的灵敏性和特异性。
47株CRE的改良Hodge实验和mCIM结果分析
47株CRE菌株产酶类型的结果分析
如图1所示:N:阴性对照肺炎克雷伯菌ATCC 700603,ETP:厄他培南,37-40为临床菌株的编号,37-40号菌株改良Hodge试验为阳性菌株。
如图2所示:MEM:美罗培南纸片,36-40为临床菌株的编号,36-40号mCIM实验均为阳性菌株。
如图3所示:MEM:美罗培南纸片,36-40为临床菌株的编号,39号eCIM实验为阳性且抑菌圈直径为25mm,而39号mCIM抑菌圈直径为6mm,39号菌株为金属酶检测阳性菌株(d39=deCIM39-dmCIM39=25-6=19mm)。
如图4所示:mCIM阳性,eCIM阴性,表明受试菌株丝氨酸碳青霉烯酶阳性。
如图5所示mCIM阳性,eCIM阳性,表明受试菌株金属酶阳性。
如图6所示mCIM阴性,eCIM不解释,表明受试菌株碳青霉烯酶阴性。
Claims (5)
1.CRE菌株碳青霉烯酶检测方法的性能评估方法,其特征在于,包括以下步骤;
步骤1:VITEK2鉴定的亚胺培南耐药菌株复苏后用加热煮沸法提取DNA模板,检测5个碳青霉烯酶基因,PCR扩增产物在琼脂糖凝胶电泳仪上进行电泳确认;
步骤2:改良Hodge试验以厄他培南为检测底物,检测菌株划线与大肠埃希菌抑菌环交叉处细菌有增强生长的趋势判为阳性,即产碳青霉烯酶;
步骤3:mCIM和eCIM:取2管2mL TSB肉汤,一管为mCIM试验管,另一管加入20μL 0.5mol/L EDTA溶液作为eCIM试验管,用1μL接种环刮取满环血平板上过夜培养的受试菌菌株,分别加入上述2管TSB肉汤中(mCIM管和eCIM管),震荡混匀;得到得到2管含有受试菌的菌液;
步骤4:将美罗培南纸片(10μg)浸入菌液中;
步骤5:制备0.5麦氏浊度单位大肠埃希菌ATCC 25922菌悬液并均匀涂布M-H琼脂平板,干燥;
步骤6:用10μL接种环将步骤4得到的美罗培南纸片取出并挤去多余菌液,贴至上述M-H琼脂板上,同一受试菌mCIM和eCIM试验管中的美罗培南纸片贴于同一M-H平板,温育,量取抑菌圈直径;
步骤7:mCIM结果判断:
碳青霉烯酶阳性:美罗培南抑菌圈直径为6-15mm或直径为16-18mm但抑菌圈内有散在菌落;碳青霉烯酶阴性:抑菌圈直径≥19mm;碳青霉烯酶中性:抑菌圈直径为16-18mm,或直径为≥19mm但抑菌圈内有散在菌落;
eCIM结果判断:金属酶阳性:与mCIM结果相比,美罗培南抑菌圈直径≥5mm(如mCIM=6mm,eCIM=15mm,抑菌圈直径之差为9mm);金属酶阴性:与mCIM结果相比,美罗培南抑菌圈直径≤4mm(如mCIM=6mm,eCIM=8mm,抑菌圈直径之差为2mm)。
2.根据权利要求1所述的CRE菌株碳青霉烯酶检测方法的性能评估方法,其特征在于,所述的EDTA溶液终浓度为5mmol/L。
3.根据权利要求1所述的CRE菌株碳青霉烯酶检测方法的性能评估方法,其特征在于,所述的步骤4中将美罗培南纸片浸入菌液中,35℃温育4h。
4.根据权利要求1所述的CRE菌株碳青霉烯酶检测方法的性能评估方法,其特征在于,所述的步骤5中干燥3~10min。
5.根据权利要求1所述的CRE菌株碳青霉烯酶检测方法的性能评估方法,其特征在于,所述的步骤6中35℃温育18~24h。
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