CN112034021B - 检测大肠杆菌内氟含量的方法 - Google Patents
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Abstract
本发明属于微生物检测领域,涉及一种检测大肠杆菌内氟含量的方法,包括:1)配制含有不同浓度氟的LB液体培养基;2)在LB液体培养基中加入野生型大肠杆菌菌株并振荡培养;3)紫外检测在野生型大肠杆菌生长的OD600值;4)收集培养的菌液后离心,倒掉上清液,重悬浮再离心后倒掉上清液后再重悬浮,水浴加热,冷却后离心;5)取上清液,用氟离子选择性电极进行测定,得到氟离子浓度C;6)计算得到野生型大肠杆菌内累积的氟含量。本发明提供了一种检测大肠杆菌内氟含量的方法,为含氟抗菌药物的研发提供浓度参考和配比依据,解决含氟抗菌药物研发时无法分别确定药物中氟和致死成分的有效浓度的问题。
Description
技术领域
本发明属于微生物检测领域,涉及一种检测大肠杆菌内氟含量的方法。
背景技术
大肠杆菌是一种条件致病菌,在合适的条件下能够寄居在人或动物的肠道内,容易引发胃肠道感染等疾病。大肠杆菌感染的发生与流行传播是呈世界性分布并具有区域性分布的特点,尤其是患病或带菌的动物往往会成为动物来源食品污染的根源,如来自带菌牛的牛肉或奶制品,来自带菌鸡的鸡蛋或鸡肉制品。而且患病或带菌动物也可通过排泄的粪便来污染当地的草场、水源等,从而造成交叉污染,甚至会区域性爆发大肠杆菌疫情。大肠杆菌感染后症状较轻的通常只会引起腹泻,但对于免疫力本身就低下的婴幼儿、年长者及患者,严重的会引发大肠杆菌性脑膜炎、败血症等疾病。因此抗菌治疗及抗菌药物的研发十分重要。
在进行抗大肠杆菌治疗时,常用氟喹诺酮类,如诺氟沙星、氧氟沙星、洛美沙星等这类含氟的抗菌药物。含氟药物具有用量少、毒性低、药效高、代谢功能强等特点。但含氟药物的广泛使用和滥用,导致大肠杆菌对药物的敏感性逐渐降低,耐药大肠杆菌出现并快速传播着。大肠杆菌的耐药机制主要分为两类,一类是基因突变或蛋白变化导致的抗生素作用位点改变或者是大肠杆菌自身产生了破坏抗生素结构的酶类,这一类机制会使得抗生素中的致死成分失效或活性减弱;另一类机制是与抗生素摄入有关的,大肠杆菌发生突变导致的某种特异膜孔蛋白丢失或发生形状或数量的改变,致使抗生素的通透性降低,或者是大肠杆菌自身的主动外排系统将药物选择性或非选择性地排出了细胞,这一类机制会使得进入细胞的药物量减少,因此抑菌作用减弱。
含氟药物中的氟主要发挥增加药物脂溶性、增强对组织细胞的穿透力的作用,能够很大程度上帮助增加药物中致死成分的抗菌谱和杀菌效果,因此适当增加药物中氟的比例能够有效抑制耐药大肠杆菌的生长。但也不能为了抑菌效果一味增大氟的比例,因为氟摄入量超标会增加氟中毒的风险,因此药物中残留的氟如果过多也会反过来伤害人体。在药物研发时,常利用外源施加含氟药物来处理大肠杆菌,通过判断大肠杆菌的生长活性来找到抑制大肠杆菌的药物浓度。常规的药敏试验只能判断出抑菌药物的有效浓度范围,无法分别确定药物中氟和致死成分的有效浓度。但若通过测定大肠杆菌中累积的氟含量就可以得到直接发挥作用的氟成分浓度,可以帮助调整合成抗菌药物的成分配比。
发明内容
为了解决背景技术中存在的上述技术问题,本发明提供了一种检测大肠杆菌内氟含量的方法。
为实现上述目的,本发明采用如下技术方案:
一种检测大肠杆菌内氟含量的方法,所述检测大肠杆菌内氟含量的方法包括以下步骤:
1)配制含有不同浓度氟的LB液体培养基;
2)分别向步骤1)配置得到的含有不同浓度氟的LB液体培养基中加入野生型大肠杆菌菌株并振荡培养;
3)紫外检测在含有不同浓度氟的LB液体培养基中野生型大肠杆菌生长的OD600值;
4)分别收集步骤3)中培养的菌液后离心,倒掉上清液,重悬浮再离心后倒掉上清液后再重悬浮,水浴加热,冷却后离心;
5)取步骤4)中最后得到的上清液,用氟离子选择性电极进行测定,得到氟离子浓度C;
6)将步骤3)检测得到的OD600值以及步骤5)测定得到的氟离子浓度C代入如下公式,计算得到野生型大肠杆菌内累积的氟含量;
其中:
C是氟离子选择性电极测定得到的氟离子浓度,单位是ppm;
V是步骤4)中最后一次重悬浮所采用的去离子水的体积,单位mL;
OD是步骤3)检测得到野生型大肠杆菌生长的OD600值。
作为优选,本发明所采用的步骤1)的具体实现方式是:
1.1)配制LB液体培养基,所述LB液体培养基的成分及含量是:胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L;
1.2)向步骤1.1)中加入不同浓度的氟离子,配制成氟离子浓度分别为0mM、15mM、25mM、50mM的含氟的LB液体培养基;
1.3)调节pH=7.0~7.4,120℃灭菌20min,冷却至40~50℃后得到含有不同浓度氟的LB液体培养基。
作为优选,本发明所采用的步骤2)中野生型大肠杆菌菌株与含有不同浓度氟的LB液体培养基的比例是1:100~1000;所述所述步骤2)中振荡培养的条件是恒温37℃、200~220rpm下振荡培养10h。
作为优选,本发明所采用的步骤4)的具体实现方式是:
4.1)分别收集步骤3)中培养的菌液后离心,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.2)加入去离子水重悬浮底部的菌块,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.4)加入去离子水重悬浮底部的菌块,在100℃下水浴加热30min,待冷却至室温后,在25℃以及4000r/min的条件下离心15min。
作为优选,本发明所采用的步骤4.2)和步骤4.4)之间还包括:
4.3)重复步骤4.2)两次。
作为优选,本发明所采用的步骤5)的具体实现方式是:
取步骤4.4)中得到的上清液,以1:1的比例加入氟离子强度缓冲液(TISABⅡwithCDTA/Thermo Orion 940909),充分混合后,用氟离子选择性电极进行测定,得到氟离子浓度C。
作为优选,本发明所采用的紫外分光光度计型号是MAPADA P4。
作为优选,本发明所采用的氟离子选择性电极型号是Thermo Orin 9609BNWP。
本发明的优点是:
本发明提供了一种检测大肠杆菌内氟含量的方法,该方法包括:1)配制含有不同浓度氟的LB液体培养基;2)向含氟液体LB中加入大肠杆菌野生型菌株菌液并振荡培养;3)紫外检测含氟培养液中大肠杆菌生长的OD600值;4)收集培养的菌液,分装并离心;5)倒掉上清液,重悬浮再离心;6)重复重悬浮再离心两次;7)倒掉上清液,重悬浮,水浴加热,冷却后离心;8)取上清液,用氟离子选择性电极进行测定;9)将数据代入公式,计算得出大肠杆菌内累积的氟含量。本发明目的是提供一种测定大肠杆菌内氟含量的方法,为含氟抗菌药物的研发提供浓度参考和配比依据,解决含氟抗菌药物研发时无法分别确定药物中氟和致死成分的有效浓度的问题。本发明提供的测定大肠杆菌内氟含量的方法首先可以帮助判断出变异耐药大肠杆菌的耐药机制,如果在相同浓度药物中培养的变异菌株的氟累积量低于普通野生型大肠杆菌,那么就可以确定菌株的耐药性变化是通过减少抗生素摄入使得进入细胞的药物量减少来实现的,就可以适当改变药物比例、增大抗生素的通透性,从而调整合成抗菌药物成分配比。通过该方法来指导合成药物的生产,可以使药物中氟和致死成分的配比更加合理和高效,避免氟残留量过高和药物浪费;本发明相较于传统的化学检测和仪器分析方法,前期处理操作更简单,易于学习和培训。
附图说明
图1是不同浓度氟处理下大肠杆菌培养10h后的OD600的曲线图;
图2是不同浓度氟处理下大肠杆菌内累积的氟含量的柱状图;
图3是一定浓度氟处理下野生型和突变型大肠杆菌内累积的氟含量的对比图。
具体实施方式
本发明提供了一种检测大肠杆菌内氟含量的方法,该方法包括以下步骤:
1)配制含有不同浓度氟的LB液体培养基,具体是:
1.1)配制LB液体培养基,LB液体培养基的成分及含量是:胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L;
1.2)向步骤1.1)中加入不同浓度的氟离子,配制成氟离子浓度分别为0mM、15mM、25mM、50mM的含氟的LB液体培养基;
1.3)调节pH=7.0~7.4,120℃灭菌20min,冷却至40~50℃后得到含有不同浓度氟的LB液体培养基。
2)分别向步骤1)配置得到的含有不同浓度氟的LB液体培养基中加入野生型大肠杆菌菌株(大肠杆菌BL21(DE3))并振荡培养;野生型大肠杆菌菌株与含有不同浓度氟的LB液体培养基的比例是1:100~1000;步骤2)中振荡培养的条件是恒温37℃、200~220rpm下振荡培养10h。
3)紫外检测在含有不同浓度氟的LB液体培养基中野生型大肠杆菌生长的OD600值;紫外分光光度计型号是MAPADA P4。
4)分别收集步骤3)中培养的菌液后离心,倒掉上清液,重悬浮再离心后倒掉上清液后再重悬浮,水浴加热,冷却后离心,具体操作是:
4.1)分别收集步骤3)中培养的菌液后离心,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.2)加入去离子水重悬浮底部的菌块,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.3)重复步骤4.2)两次;
4.4)加入去离子水重悬浮底部的菌块,在100℃下水浴加热30min,待冷却至室温后,在25℃以及4000r/min的条件下离心15min。
5)取步骤4.4)中得到的上清液,以1:1的比例加入氟离子强度缓冲液,充分混合后,用氟离子选择性电极进行测定,得到氟离子浓度C,氟离子选择性电极型号是ThermoOrin9609BNWP。
6)将步骤3)检测得到的OD600值以及步骤5)测定得到的氟离子浓度C代入如下公式,计算得到野生型大肠杆菌内累积的氟含量;
其中:
C是氟离子选择性电极测定得到的氟离子浓度,单位是ppm;
V是步骤4)中最后一次重悬浮所采用的去离子水的体积,单位mL;
OD是步骤3)检测得到野生型大肠杆菌生长的OD600值。
实施例1:大肠杆菌生长OD600值的测定
1)配制LB液体培养基:胰蛋白胨10g、酵母提取物5g、氯化钠10g,加入到1000mL去离子水中,充分混合;配制800mMNaF母液;分别称取200mL液体LB,向其中加入NaF母液,配制成氟离子浓度分别为0mM、15mM、25mM、50mM的含氟的LB液体培养基,并调节pH为7.0,120℃灭菌20min,冷却至40℃;
2)向步骤1)中的含氟液体LB中分别加入2mL大肠杆菌野生型菌株菌液,菌液与含氟液体LB的比例为1:100,恒温37℃、220rpm下振荡培养10h;
3)采用紫外分光光度计(MAPADA P4)于波长600nm处检测步骤2)中0mM、15mM、25mM、50mM的含氟培养液中大肠杆菌生长的OD600值,分别为1.737、1.117、0.716、0.185(参见图1),用于代入公式中计算大肠杆菌中累积的氟含量。
实施例2:大肠杆菌氟累积量的测定与计算
1)收集培养的菌液,分装在50mL离心管中,25℃、4000r/min下离心15min;
2)倒掉离心后的上清液,再加入30mL去离子水重悬浮底部的菌块,再25℃、4000r/min下离心15min;
3)重复步骤2)两次;
4)倒掉步骤3)中离心后的上清液,加入30mL去离子水,重悬浮,在100℃下水浴加热30min,待冷却至室温后,25℃、4000r/min下离心15min;
5)取步骤4)中离心后的上清液,以1:1的比例加入15mL氟离子强度缓冲液,充分混合后,用氟离子选择性电极(Thermo Orin 9609BNWP)进行测定,得到氟离子浓度C;
6)将得到的数据代入公式,计算得出0mM、15mM、25mM、50mM的含氟培养液中大肠杆菌内累积的氟含量,参见图2,结果分别为0.00146mg·OD-1、0.00243mg·OD-1、0.00524mg·OD-1、0.587mg·OD-1,说明每生长单位的大肠杆菌内的氟含量分别为0.00146mg、0.00243mg、0.00524mg、0.587mg。
本实施方式可以利用大肠杆菌生长OD600值和氟离子选择性电极直接测定并计算出大肠杆菌中累积的氟含量。本发明提供的测定大肠杆菌内氟含量的方法首先可以帮助判断出变异耐药大肠杆菌的耐药机制,如果在相同浓度药物中培养的变异菌株的氟累积量低于普通野生型大肠杆菌,那么就可以确定菌株的耐药性变化是通过减少抗生素摄入使得进入细胞的药物量减少来实现的,就可以适当改变药物比例、增大抗生素的通透性,从而调整合成抗菌药物成分配比。通过该方法来指导合成药物的生产,可以使药物中氟和致死成分的配比更加合理和高效,避免氟残留量过高和药物浪费。
1)分别称取200mL液体LB,向其中加入NaF母液,配制成一定浓度的含氟的LB液体培养基,并调节pH为7.0,120℃灭菌20min,冷却至40℃;
2)分别加入2mL野生型大肠杆菌菌液和突变型大肠杆菌菌液,菌液与含氟液体LB的比例为1:100,恒温37℃、220rpm下振荡培养10h;
3)采用紫外分光光度计(MAPADA P4)于波长600nm处检测步骤2)中野生型和突变型大肠杆菌生长的OD600值,分别为0.008、0.019,用于代入公式中计算大肠杆菌中累积的氟含量。
4)收集培养的菌液,分装在50mL离心管中,25℃、4000r/min下离心15min;
5)倒掉离心后的上清液,再加入30mL去离子水重悬浮底部的菌块,再25℃、4000r/min下离心15min;
6)重复步骤5)两次;
7)倒掉步骤6)中离心后的上清液,加入30mL去离子水,重悬浮,在100℃下水浴加热30min,待冷却至室温后,25℃、4000r/min下离心15min;
8)取步骤7)中离心后的上清液,以1:1的比例加入15mL氟离子强度缓冲液,充分混合后,用氟离子选择性电极(Thermo Orin 9609BNWP)进行测定,得到氟离子浓度C;
9)将得到的数据代入公式,计算得出含氟培养液中野生型和突变型大肠杆菌内累积的氟含量,参见图3,结果分别为0.149mg·OD-1、0.0198mg·OD-1,说明每生长单位的野生型和突变型大肠杆菌内的氟含量分别为0.149mg、0.0198mg。
突变型大肠杆菌是过表达CsFEX型大肠杆菌,该过表达CsFEX型大肠杆菌的获得方式是:
基于保守的crcB结构域,从Pan等(2016)的转录组数据(PRJNA315669)中筛选出CsFEX基因,后使用引物(CsFEX-F/R)通过PCR扩增获得CsFEX的开放阅读框(ORF),含有1443bp,随后纯化扩增产物并将该基因克隆到克隆载体pDONR201中。
将CsFEX的ORF连接到表达载体pET-28a后,把pET-CsFEX重组质粒和空载体(pET-28a)转化到大肠杆菌BL21(DE3)感受态细胞中。将转化后的菌液涂布于含50μg/mL卡那霉素的LB固体培养基上,恒温37℃下静置培养12h,长出单菌落,得到过表达CsFEX型大肠杆菌E.coli BL21(DE3)/pET 28a-CsFEX。
本例中用到的突变型大肠杆菌菌株是一种特殊的氟转运蛋白过表达型菌株,能够有效减少氟化物进入细胞,结果得到该菌株内的氟含量比普通野生型的更低,也印证了这一特点。因此为了抑制这类突变型大肠杆菌的生长,需要调整抗菌药物中氟的比例来增大药物的通透性。
Claims (8)
1.一种检测大肠杆菌内氟含量的方法,其特征在于:所述检测大肠杆菌内氟含量的方法包括以下步骤:
1)配制含有不同浓度氟的LB液体培养基;
2)分别向步骤1)配置得到的含有不同浓度氟的LB液体培养基中加入野生型大肠杆菌菌株并振荡培养;
3)紫外检测在含有不同浓度氟的LB液体培养基中野生型大肠杆菌生长的OD600值;
4)分别收集步骤3)中培养的菌液后离心,倒掉上清液,重悬浮再离心后倒掉上清液后再重悬浮,水浴加热,冷却后离心;
5)取步骤4)中最后得到的上清液,用氟离子选择性电极进行测定,得到氟离子浓度C;
6)将步骤3)检测得到的OD600值以及步骤5)测定得到的氟离子浓度C代入如下公式,计算得到野生型大肠杆菌内累积的氟含量;
其中:
C是氟离子选择性电极测定得到的氟离子浓度,单位是ppm;
V是步骤4)中最后一次重悬浮所采用的去离子水的体积,单位mL;
OD是步骤3)检测得到野生型大肠杆菌生长的OD600值。
2.根据权利要求1所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤1)的具体实现方式是:
1.1)配制LB液体培养基,所述LB液体培养基的成分及含量是:胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L;
1.2)向步骤1.1)中加入不同浓度的氟离子,配制成氟离子浓度分别为0mM、15mM、25mM、50mM的含氟的LB液体培养基;
1.3)调节pH=7.0~7.4,120℃灭菌20min,冷却至40~50℃后得到含有不同浓度氟的LB液体培养基。
3.根据权利要求2所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤2)中野生型大肠杆菌菌株与含有不同浓度氟的LB液体培养基的比例是1:100~1000;所述步骤2)中振荡培养的条件是恒温37℃、200~220rpm下振荡培养10h。
4.根据权利要求3所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤4)的具体实现方式是:
4.1)分别收集步骤3)中培养的菌液后离心,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.2)加入去离子水重悬浮底部的菌块,在25℃以及4000r/min的条件下离心15min,倒掉上清液;
4.4)加入去离子水重悬浮底部的菌块,在100℃下水浴加热30min,待冷却至室温后,在25℃以及4000r/min的条件下离心15min。
5.根据权利要求4所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤4.2)和步骤4.4)之间还包括:
4.3)重复步骤4.2)两次。
6.根据权利要求5所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤5)的具体实现方式是:
取步骤4.4)中得到的上清液,以1:1的比例加入氟离子强度缓冲液,充分混合后,用氟离子选择性电极进行测定,得到氟离子浓度C。
7.根据权利要求1-6任一项所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤3)中所采用的紫外分光光度计型号是MAPADA P4。
8.根据权利要求7所述的检测大肠杆菌内氟含量的方法,其特征在于:所述步骤5)中所采用的氟离子选择性电极型号是Thermo Orin 9609BNWP。
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