CN111304343A - 一种海水浴场中磺胺类抗生素抗性基因的定量检测方法 - Google Patents
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Abstract
本发明公开了一种海水浴场中磺胺类抗生素抗性基因的定量检测方法,包括海水浴场水样和沉积物的采集并低温保存,对采集样品进行DNA提取和纯化后利用PCR扩增抗生素抗性基因的DNA片段,克隆该DNA片段到宿主细胞构建标准质粒,制备目标DNA标准品的荧光定量PCR标准曲线,根据标准品PCR标准曲线和纯化后样品的CT值计算样品中抗生素抗性基因的含量,本发明方法步骤简单、操作方便,省略了样品DNA序列分析步骤,根据PCR标准曲线和样品的CT值得到样品水体和沉积物中抗生素抗性基因的含量,适用范围广。
Description
技术领域
本发明属于检测技术领域,具体涉及一种海水浴场中磺胺类抗生素抗性基因的定量检测方法。
背景技术
随着滨海旅游资源开发的日益加剧以及海岸带人口的不断增加,海水浴场已逐步成为抗生素抗性基因传播扩散的重要媒介之一。抗生素抗性基因会随雨水冲刷、潮汐和海流带入近岸的海滨旅游区,通过菌种间遗传物质的传递,抗生素抗性基因有可能被传入海水环境中的人体病原菌。由于海滩娱乐水域承载了大量的游客,这些抗生素抗性基因可通过海水吞咽引发游泳人群致命性肠道疾病,因而大大增加了人体耐药病原菌疾病发生和流行的可能性。我国海岸线漫长,滨海旅游资源丰富,海水浴场众多,因而通过海水浴场传播抗生素抗性基因已逐步成为威胁公众健康的重要问题之一。
海洋环境中的抗性基因主要源于污水处理厂、抗生素生产厂、养殖场和水产养殖的排放。抗生素抗性基因已在海洋不同环境介质中被检出。目前,渤海、黄海及河口等海洋环境不同介质中都检出较高丰度的磺胺类抗性基因(sul1和sul2)。磺胺类抗生素是一类具有广谱抗菌活性的人畜共用抗菌剂,被广泛用于医疗、畜牧和水产养殖等领域。因而,磺胺类抗生素引发的磺胺类抗性基因污染日益严重。然而我国对海水浴场中抗性基因分布研究起步较晚,关注较少,目前尚未建立统一的检测方法来测定海水浴场中的抗生素抗性基因。因此,海水浴场中磺胺类抗生素抗性基因检测方法的建立对于研究抗生素抗性基因在海水浴场基质中的传播扩散及人体健康风险评估等方面的研究具有重要意义。
因此,为加强海水浴场水质监测和环境保护、规范公共卫生标准和污水排放政策、保障游客的生命安全,亟需开发一种海水浴场中磺胺类抗生素抗性基因的定量检测方法。
发明内容
本发明的目的在于提供一种测定海水浴场中磺胺类抗生素抗性基因的定量检测方法。
为实现上述技术目的,本发明采用如下技术方案:
一种海水浴场中磺胺类抗生素抗性基因的定量检测方法,包括采集水体和沉积物样本、样本总DNA提取、目标DNA标准品制备及荧光定量PCR检测,所述抗性基因为磺胺类抗生素抗性基因sul1和磺胺类抗生素抗性基因sul2;所述荧光定量PCR反应体系为:
TB Green Premix 10μl、
上游引物(10μM)0.4μl;
下游引物(10μM)0.4μl;
ROX II 0.4μl;
ddH2O 6.8μl;
DNA模板2μl,一共20μl。进一步的,所述上游引物和下游引物序列为:
Sul1:
上游引物:CGCACCGGAAACATCGCTGCAC;
下游引物:TGAAGTTCCGCCGCAAGGCTCG。
Sul2:
上游引物:TCCGGTGGAGGCCGGTATCTGG;
下游引物:CGGGAATGCCATCTGCCTTGAG。
sul1反应条件为95℃预变性15min;95℃变性15s→65℃退火30s→72℃延伸34s,循环次数40次。
sul2反应条件为95℃预变性15min;95℃变性15s→57.5℃退火30s→72℃延伸34s,循环次数50次。
然后添加熔解曲线,以确认引物的特异性良好。
进一步的,所述目标DNA标准品制备过程包括:
(1)通过普通PCR反应扩增出抗生素抗性基因的DNA片段;
(2)切胶回收抗生素抗性基因的PCR产物;
(3)将回收的PCR产物与克隆载体连接成环状质粒,转化至感受态细胞进行克隆;
(4)通过蓝白实验筛选阳性克隆子,分离纯化后提取质粒;
(5)梯度稀释标准质粒制备荧光定量PCR标准曲线。
其中,所述步骤(1)中,以抗生素抗性基因阳性菌株所提取的基因组DNA为模板扩增出200bp以下的DNA片段。
所述步骤(5)中,用Easy Dilution将所提质粒稀释为梯度浓度的标准DNA溶液,以建立实时荧光定量PCR的标准曲线。
本发明通过荧光定量PCR技术对海水浴场水体和沉积物中磺胺类抗生素抗性基因进行了定量检测,可辅助了解海水浴场自然水体和沉积物中磺胺类抗生素抗性基因的含量水平和分布特征。采用本发明的方法进行抗生素抗性基因Sul1和Sul2的检测,省略了样品DNA序列分析步骤,适用范围广,PCR扩增效率高,测量准确度高。
附图说明
图1是sul1荧光定量PCR的扩增曲线;
图2是sul1荧光定量PCR的标准曲线;
图3是sul2荧光定量PCR的扩增曲线;
图4是sul2荧光定量PCR的标准曲线;
图5是各水体样本中sul1含量;
图6是各沉积物样本中sul1含量;
图7是各水体样本中sul2含量;
图8是各沉积物样本中sul2含量。
具体实施方式
本实施例具体说明测定海水浴场中磺胺类抗生素抗性基因sul1和sul2的定量检测方法。
步骤一:海水浴场水样和沉积物的采集方法
水样:用灭菌的5L聚乙烯桶采集水样。采集时取表层水为宜,每个采样点采集3桶为3个样品平行,标上编号、采集地点、采集时间。采集后马上将水样放入盛有冰块的保温箱中,及时带回实验室处理。
沉积物:表层沉积物用灭菌的采样器采集,放入无菌的自封袋中。采集后马上将样品放入盛有冰块的保温箱中,及时带回实验室处理。
步骤二:样品运输与保存
样品采集后应马上放入4-10℃的冰盒中保存,2h内运回实验室24h内进行分析测定。如不能立即开展分析测定工作,应把水样保存在4-10℃暗冷处,并尽快进行分析测定,水样过0.45μm醋酸纤维滤膜,并将滤膜放入50ml离心管,并在管壁上记录过膜水样体积。若当天无法提取DNA,将其保存在-20℃的冰箱中。固体样品应在-20℃以下存放,以防DNA降解。
步骤三:样品总DNA提取
水样过膜后,用高压灭菌的剪刀把滤膜剪碎,并用DNA Power Water Total DNAIsolation Kit提取试剂盒(Qiagen),按照试剂盒说明书操作步骤进行提取和纯化样品总DNA;根据固体样品类型,称取0.5g冷冻干燥并研磨后的样品,使用DNA Power Soil TotalDNA Isolation Kit提取试剂盒(Qiagen)说明书操作步骤进行提取和纯化样品总DNA。
步骤四:抗生素抗性基因标准质粒的构建和标准曲线的制备
以耐药基因阳性菌株所提基因组DNA为模板,利用PCR技术扩增抗生素抗性基因的DNA片段,克隆该DNA片段到宿主细胞构建标准质粒,制备荧光定量PCR的标准曲线。
具体实验步骤为:
1.通过普通PCR反应扩增出抗生素抗性基因的DNA片段。
2.切胶回收抗生素抗性基因的PCR产物。
3.回收后的PCR产物与PMD-19T载体连接成环状质粒,转化进E.coli DH5α感受态细胞进行克隆。添加X-gal以及IPTG后,将感受态细胞菌液均匀涂布于LB-氨苄培养基上过夜培养。
4.通过蓝白实验筛选阳性克隆子,分离纯化后提取质粒。
5.梯度稀释标准质粒制备荧光定量PCR标准曲线。
利用PCR技术,以抗生素抗性基因阳性菌株所提基因组DNA为模板扩增出200bp以下的DNA片段。
PCR反应体系如下:
Premix Taq 12.5μL、
上下游引物(10μM)各0.2μL、
DNA模板2μL、
然后使用ddH2O补充至25μL体系。
sul1反应条件为95℃预变性5min;95℃变性15s→56℃退火30s→72℃延伸30s,循环次数40次;最后72℃延伸7min。
sul2反应条件为95℃预变性5min;95℃变性15s→61℃退火30s→72℃延伸30s,循环次数40次;最后72℃延伸7min。
将目标基因的PCR产物用琼脂糖凝胶回收试剂盒进行切胶回收,具体操作参照试剂盒说明书。将纯化后的目标基因的PCR产物连接到PMD-19T载体上。将连接了耐药基因DNA片段的载体转化进入E.coli DH5α感受态细胞,通过蓝白实验筛选出阳性克隆子,用目标基因的引物和PMD-19T载体的引物分别进行PCR反应对克隆子进行进一步确认。
经确认后的阳性克隆子在LB培养基中37℃下过夜培养,用质粒提试剂盒提取阳性克隆子中已连接抗生素抗性基因的载体质粒。具体实验操作见质粒提试剂盒说明书。
测定所提载体质粒的核酸浓度,根据质粒大小(PMD-19T载体与耐药基因PCR产物碱基对之和)计算出目标耐药基因的拷贝数(copies/μL),计算公式为:
C为所提质粒的浓度,L为所提质粒碱基对的数目(PMD-19T载体碱基对数2682bp加上目标耐药基因PCR产物的碱基对数)。
根据计算结果,用Easy Dilution把所提质粒稀释成108、107、106、105、104、103、102copies/μL的标准DNA溶液,以此建立实时荧光定量PCR的标准曲线。
用目标基因标准质粒的梯度溶液为DNA模板,对其进行荧光定量PCR扩增,得到各个浓度的CT值,并确认实时定量PCR的扩增曲线和融解曲线。以copies/mL(g)为纵坐标,CT值为横坐标,得到标准曲线和一元线性方程。
本实施例中sul1标准曲线R 2=0.998,PCR扩增效率为96.789%;sul1的荧光定量PCR扩增曲线和标准曲线分别如图1、图2所示。sul2标准曲线R 2=0.999,PCR扩增效率(Amplification Efficiency)为96.932%;sul2的荧光定量PCR扩增曲线和标准曲线分别如图3、图4所示。结果表明扩增效率超过90%,线性相关良好,能够准确的反映目标产物的扩增,可用于计算基因拷贝数。
步骤五:抗生素抗性基因荧光定量PCR检测方法
反应体系:采用SYBRGreen染料法,
实时荧光定量PCR反应体系为
TB Green Premix 10μl,
PCR上游引物(10μM)0.4μL,
PCR下游引物(10μM)0.4μL,
ROX II 0.4μl,
ddH2O 6.8μl
DNA模板2μl,一共20μl。
所述上游引物和下游引物序列为:
Sul1:
上游引物:CGCACCGGAAACATCGCTGCAC;
下游引物:TGAAGTTCCGCCGCAAGGCTCG.
Sul2:
上游引物:TCCGGTGGAGGCCGGTATCTGG;
下游引物:CGGGAATGCCATCTGCCTTGAG。
根据样品数量配置反应溶液体系,混合均匀后分装18μL溶液到每管中,然后分别加入提取的样品总DNA溶液2μL做模板。每个样品设置3个平行。
反应程序为:sul1反应条件为95℃预变性15min;95℃变性15s→65℃退火30s→72℃延伸34s,循环次数40次。
sul2反应条件为95℃预变性15min;95℃变性15s→57.5℃退火30s→72℃延伸34s,循环次数50次。然后添加融解曲线,以确认引物的特异性良好。
样品检测:对纯化后的样品基因组DNA进行荧光定量PCR扩增,并确认其扩增曲线和融解曲线。根据标准曲线和样品的CT值得到浴场海水中抗生素抗性基因的含量和沉积物样品中抗生素抗性基因的含量(copies/mLDNA),通过数学计算出沉积物样品中耐药基因的含量(copies/g)。各水体、沉积物样本中sul1含量分别如图5、图6所示;各水体、沉积物样本中Sul2含量分别如图7、图8所示。结果显示样品中sul1、sul2的检出率为100%,说明sul1、sul2广泛存在于海水浴场中,且抗性基因在水体和沉积物中的含量差异较大,两者之间相差约1个数量级;此外,由于星海浴场受污染程度比棒槌岛浴场严重,导致星海浴场中sul1、sul2含量普遍高于棒槌岛浴场中sul1、sul2含量。
上述实施例仅为本发明的优选实施方式之一,不应当用于限制本发明的保护范围,但凡在本发明的主体设计思想和精神上作出的毫无实质意义的改动或润色,其所解决的技术问题仍然与本发明一致的,均应当包含在本发明的保护范围之内。
Claims (5)
1.一种海水浴场中磺胺类抗生素抗性基因的定量检测方法,包括采集水体和沉积物样本和样本总DNA提取,其特征在于,还包括制备目标DNA标准品和对样品基因组DNA进行荧光定量PCR检测,
所述耐药基因为磺胺类抗生素抗性基因sul1和磺胺类抗生素抗性基因sul2;
所述荧光定量PCR反应体系为:
TB Green Premix 10μl,
PCR上游引物(10μM)0.4μL,
PCR下游引物(10μM)0.4μL,
ROX II 0.4μl,
ddH2O 6.8μl
DNA模板2μl,一共20μl。
sul1反应条件为95℃预变性15min;95℃变性15s→65℃退火30s→72℃延伸34s,循环次数40次。
sul2反应条件为95℃预变性15min;95℃变性15s→57.5℃退火30s→72℃延伸34s,循环次数50次。
2.根据权利要求1所述的海水浴场中磺胺类抗生素抗性基因的定量检测方法,其特征在于,目标DNA标准品制备过程包括:
(1)通过PCR反应扩增出耐药基因的DNA片段;
(2)切胶回收耐药基因的普通PCR产物;
(3)将回收的普通PCR产物与克隆载体连接成环状质粒,转化至感受态细菌进行克隆;
(4)通过蓝白实验筛选阳性克隆子,分离纯化后提取质粒;
(5)梯度稀释标准质粒制备荧光定量PCR标准曲线。
3.根据权利要求2所述的海水浴场中磺胺类抗生素抗性基因的定量检测方法,其特征在于,所述步骤(1)中,以耐药基因阳性菌株所提取的基因组DNA为模板扩增出200bp以下的DNA片段。
4.根据权利要求1或2所述的海水浴场中磺胺类抗生素抗性基因的定量检测方法,其特征在于,
所述上游引物和下游引物序列为:
sul1:上游引物:CGCACCGGAAACATCGCTGCAC;
下游引物:TGAAGTTCCGCCGCAAGGCTCG;
sul2:上游引物:TCCGGTGGAGGCCGGTATCTGG;
下游引物:CGGGAATGCCATCTGCCTTGAG。
5.根据权利要求2所述的海水浴场中磺胺类抗生素抗性基因的定量检测方法,其特征在于,所述步骤(5)中用Easy Dilution将所提质粒稀释为梯度浓度的标准DNA溶液,以建立实时荧光定量PCR的标准曲线。
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