CN106167821A - 一种金黄色葡萄球菌crispr位点检测试剂盒及检测方法 - Google Patents

一种金黄色葡萄球菌crispr位点检测试剂盒及检测方法 Download PDF

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CN106167821A
CN106167821A CN201610426983.4A CN201610426983A CN106167821A CN 106167821 A CN106167821 A CN 106167821A CN 201610426983 A CN201610426983 A CN 201610426983A CN 106167821 A CN106167821 A CN 106167821A
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staphylococcus aureus
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杨海燕
刘静
邵富叶
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Abstract

本发明公开了一种金黄色葡萄球菌CRISPR位点检测试剂盒及检测方法，包含针对金黄色葡萄球菌的三个CRISPR位点基因序列设计的三对特异性引物。本发明还公开了一种金黄色葡萄球菌CRISPR位点检测方法，利用三对特异性引物能够从样品基因组DNA中扩增出相应的CRISPR片段。本发明提供的试剂盒和检测方法操作简单，敏感性和特异性高，检测费用低，具有良好的推广应用价值。

Description

一种金黄色葡萄球菌CRISPR位点检测试剂盒及检测方法
技术领域
本发明属于微生物检测技术领域，特别是涉及一种金黄色葡萄球菌CRISPR位点检测试剂盒及检测方法。
背景技术
金黄色葡萄球菌是人类一种重要的致病菌，隶属于革兰染色阳性球菌，可以引起多种疾病，如心内膜炎，肺炎，中毒性休克综合征等。近些年来，随着金黄色葡萄球菌耐药率和耐药程度的不断升高，金黄色葡萄球菌感染已经成为严重的健康问题。成簇的规律间隔的短回文重复序列（Clustered regularly interspaced short palindromic repeats，CRISPR）是近年发现的针对噬菌体等外源基因的细菌免疫系统，能够有效地抵制噬菌体和外界各种基因元件对其造成的干扰，从而抑制水平基因转移（Horizontal Gene Transfer，HGT）。CRISPR广泛存在于细菌和古细菌中，是由一段不连续的重复序列（repeat，R），和插入其中的间隔序列（spacer，S）以及一系列cas蛋白组成。重复序列比较保守（包括长度和碱基序列），大多具有回文结构，可以形成稳定的高度保守的二级结构。间隔序列由26-72个碱基组成，序列高度可变，有研究表明间隔序列来源于外源核酸物质（质粒和噬菌体等）。目前CRISPR在金黄色葡萄球菌中的研究主要集中在金黄色葡萄球菌中CRISPR的功能性研究，然而市面上并没有特异性检测金黄色葡萄球菌CRISPR位点的相关试剂盒。
发明内容
本发明的目的就在于克服上述不足，提供一种金黄色葡萄球菌CRISPR位点检测试剂盒及检测方法。
为达到上述目的，本发明是按照以下技术方案实施的：
一种金黄色葡萄球菌CRISPR位点检测试剂盒，包括针对金黄色葡萄球菌的CRISPR位点基因序列设计的引物，所述金黄色葡萄球菌的CRISPR位点基因序列有三种，分别为CRISPR-1，CRISPR-2和CRISPR-3，对应的引物序列如下所示：
CRISPR-1 上游引物 5’-GTAGTGATTTTTGGGAGTGG-3’,
下游引物 5’-GTGGTTAATGTGTAGGGACC-3’,
CRISPR-2 上游引物 5’-CAATGACTTGGAGTGTGA-3’,
下游引物 5’-GGTTAATGTGTAGGAACC-3’,
CRISPR-3 上游引物 5’-ACTCAATGGCATGGAGTGTGA-3’,
下游引物 5’-GGTGGTTAATGTGTAGGGACC-3’。
一种金黄色葡萄球菌CRISPR位点检测试剂盒还包括10×Tag Buffer，0.2 mMdNTP，2 mM MgCl2，0.05U/μL Tag DNA聚合酶，灭菌超纯水和DNA Marker 2000。
一种金黄色葡萄球菌CRISPR位点检测方法，包括以下步骤：
（1）以细菌样品的基因组DNA为模板，分别利用3种CRISPR位点基因序列的引物进行PCR扩增，引物序列如下所示，
CRISPR-1 上游引物 5’-GTAGTGATTTTTGGGAGTGG-3’,
下游引物 5’-GTGGTTAATGTGTAGGGACC-3’,
CRISPR-2 上游引物 5’-CAATGACTTGGAGTGTGA-3’,
下游引物 5’-GGTTAATGTGTAGGAACC-3’,
CRISPR-3 上游引物 5’-ACTCAATGGCATGGAGTGTGA-3’,
下游引物 5’-GGTGGTTAATGTGTAGGGACC-3’，
待PCR扩增结束后，得扩增产物；
（2）将所得扩增产物经凝胶电泳分析，CRISPR-1的预期扩增片段长度为330bp，CRISPR-2的预期扩增片段长度为380bp，CRISPR-3的预期扩增片段长度为387bp，得到相应大小的基因片段即为扩增成功。。
进一步的，所述PCR扩增的反应体系为：10μmol/L上游引物1μL，10μmol/L下游引物1μL，10×Tag Buffer 2.5μL，0.2 mM dNTP 0.5μL，2 mM MgCl2 2μl，0.05U/μL Taq DNA 聚合酶0.125μL，细菌样品基因组DNA 2μL，灭菌超纯水15.875μL。
进一步的，针对不同的CRISPR位点基因序列所采用的PCR扩增反应程序分别为：
CRISPR-1 94℃预变性5min；94℃变性30s，51℃退火30s，72℃延伸30s，共35个循环，72℃继续延伸10min；
CRISPR-2 94℃预变性5min；94℃变性30s，50℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min；
CRISPR-3 94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min。
进一步的，凝胶电泳的条件为：1.5%琼脂糖凝胶，电压5V/cm，时间25min。
进一步的，细菌样品的基因组DNA采用煮沸法提取。
本发明通过对CRISPR database数据库中金黄色葡萄球菌全基因组测序结果进行分析及对实验室保存的金黄色葡萄球菌检测发现，其具有上述3种CRISPR位点存在，3种CRISPR位点均有2条重复序列和1条间隔序列。针对上述3种CRISPR位点的基因序列设计上、下游引物，经PCR扩增后对产物进行凝胶电泳分析，优化实验条件，合理设计出金黄色葡萄球菌CRISPR位点检测试剂盒，并确定最佳的检测方法。
与现有技术相比，本发明的有益效果为：本发明提供的试剂盒和检测方法操作简单，敏感性和特异性高，检测费用低，具有良好的推广应用价值。
说明书附图
图1为CRISPR-1的凝胶电泳图
1号泳道为DNA marker，2号泳道为PCR扩增产物；
图2为CRISPR-2的凝胶电泳图
1号泳道为DNA marker，2号泳道为PCR扩增产物；
图3为CRISPR-3的凝胶电泳图
1号泳道为DNA marker，2号泳道为PCR扩增产物；
图4为CRISPR-1位点基因序列的分析结果；
图5为CRISPR-1位点基因序列的分析结果；
图6为CRISPR-1位点基因序列的分析结果。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步描述，在此发明的示意性实施例以及说明用来解释本发明，但并不作为对本发明的限定。
实施例1
金黄色葡萄球菌的CRISPR位点基因序列有三种，分别为CRISPR-1，CRISPR-2和CRISPR-3。根据Genebank中已发布的金黄色葡萄球菌CRISPR相关基因序列设计3种CRISPR位点引物：
CRISPR-1 上游引物 5’-GTAGTGATTTTTGGGAGTGG-3’,
下游引物 5’-GTGGTTAATGTGTAGGGACC-3’,
CRISPR-2 上游引物 5’-CAATGACTTGGAGTGTGA-3’,
下游引物 5’-GGTTAATGTGTAGGAACC-3’,
CRISPR-3 上游引物 5’-ACTCAATGGCATGGAGTGTGA-3’,
下游引物 5’-GGTGGTTAATGTGTAGGGACC-3’。
金黄色葡萄球菌CRISPR位点检测试剂盒包括针对金黄色葡萄球菌的CRISPR位点基因序列设计的引物，10×Tag Buffer，0.2 mM dNTP，2 mM MgCl2，0.05U/μL Tag DNA聚合酶，灭菌超纯水和DNA Marker 2000。
实施例2
金黄色葡萄球菌CRISPR位点的检测方法，包括以下步骤：
（1）细菌样品的基因组DNA采用煮沸法提取。
（2）以细菌样品的基因组DNA为模板，分别利用3种CRISPR位点基因序列的引物进行PCR扩增，引物序列如实施例1所示，所述PCR扩增的反应体系为：10μmol/L上游引物1μL，10μmol/L下游引物1μL，10×Tag Buffer 2.5μL，0.2 mM dNTP 0.5μL，2 mM MgCl2 2μl，0.05U/μL Taq DNA 聚合酶0.125μL，细菌样品基因组DNA 2μL，灭菌超纯水15.875μL。针对不同的CRISPR位点基因序列所采用的PCR扩增反应程序分别为：
CRISPR-1 94℃预变性5min；94℃变性30s，51℃退火30s，72℃延伸30s，共35个循环，72℃继续延伸10min；
CRISPR-2 94℃预变性5min；94℃变性30s，50℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min；
CRISPR-3 94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min。
待PCR扩增结束后，得扩增产物；
（3）将所得扩增产物经凝胶电泳分析，凝胶电泳的条件为：1.5%琼脂糖凝胶，电压5V/cm，时间25min。CRISPR-1的预期扩增片段长度为330bp，CRISPR-2的预期扩增片段长度为380bp，CRISPR-3的预期扩增片段长度为387bp，得到相应大小的基因片段即为扩增成功。
实验例
一、样品采集
试验中使用的细菌样品为金黄色葡萄球菌，2014年分离于河南郑州某医院。
二、实验方法
采用本发明的检测试剂盒对细菌样品的CRISPR位点进行检测，检测步骤如下：
（1）DNA模板的制备
采用煮沸法从细菌样品中提取全基因组DNA。从-80℃冰箱内取出细菌样品冻存管，放于4℃冰箱复温5小时，在超净台中采用无菌接种环快速蘸取菌液，以分段划线法接种于哥伦比亚血琼脂培养板上，37℃恒温箱孵育20小时，挑取血平板上单个菌落，接种于脑心浸液液体培养基中，37℃振荡培养16小时，取1mL菌液于1.5mL的Eppdorf管中，转速为12000r/min条件下离心1分钟，将上清弃去，加入超纯水100μL，震荡混匀，煮沸10min，转速为12000r/min条件下离心10分钟，取上清，即可以得到细菌样品的基因组DNA，置于-20℃贮存备用。
（2）PCR扩增
PCR扩增的反应体系：10μmol/L 上游引物1μL，10μmol/L 下游引物1μL，10×TagBuffer 2.5μL，0.2 mM dNTP 0.5μL，2 mM MgCL2 2μL，Taq DNA 聚合酶0.125μL，金黄色葡萄球菌基因组DNA模板2μL，灭菌超纯水加至25μL。所述Tag Buffer 为(NH4)2SO4溶液，引物序列分别为：
CRISPR-1 330bp 上游引物:GTAGTGATTTTTGGGAGTGG
下游引物:GTGGTTAATGTGTAGGGACC
CRISPR-2 380bp 上游引物:CAATGACTTGGAGTGTGA
下游引物:GGTTAATGTGTAGGAACC
CRISPR-3 387bp 上游引物:ACTCAATGGCATGGAGTGTGA
下游引物:GGTGGTTAATGTGTAGGGACC
PCR扩增的反应程序如下表所示：
CRISPR位点 循环参数 循环次数
CRISPR-1 94 ℃ 30 s， 51 ℃30 s， 72 ℃ 30 s 35
CRISPR-2 94 ℃ 30 s， 50 ℃30 s， 72 ℃ 45 s 35
CRISPR-3 94 ℃ 30 s， 55 ℃30 s， 72 ℃ 45 s 35
（3）PCR扩增产物分析
取5 μL PCR产物与1μL 6×loading buffer混匀，进行电泳，采用1×TAE电泳缓冲液，浓度为1.5%的琼脂糖凝胶，电压5V/cm，电泳时间为25min，电泳结束后，取出凝胶并采用使用凝胶成像仪观察结果并拍照保存。
三、实验结果
（1）PCR扩增产物凝胶电泳鉴定
PCR扩增3种CRISPR位点产物的电泳结果见图1、图2、图3。根据引物设计推断，CRISPR-1的扩增片段长度为330bp，CRISPR-2的扩增片段长度为380bp，CRISPR-3的扩增片段长度为387bp。从图1-3中可以看出，PCR扩增产物的长度与预期长度基本符合，证实PCR扩增3种CRISPR位点成功。
（2）PCR扩增结果测序鉴定
将PCR扩增产物交由上海生工生物工程股份有限公司进行测序，测序结果如下所示：
CRISPR-1的序列为
CACGGTGATAGAGCACGGTCTTTTATTCTTTGTCATTAGCCACAGCTATTGTGTACTTAAAAATAGGAATGCATGAGTGCAACTCATGCATAAGAAATACTAATTTCTAAAGAAAAAGTATTTCTTTATGTTGGGGCCCACCCCAACTTGCATTGTTTGTAGAATTTCTTTTCGAAATTCTCTGTGTTGGGGCCCCGCCAACTTGCATTGCCTGTAGAATTTCTTTTCGAAATTCTTTATGTTGGGGCCCCGCCAACTAATTACAATATATCATTGTAGAGCTTAGGTCATTGATTTTTGGCTCGGACTTTTATGA
CRISPR-2的序列为
ATAACCTAAGATTTATTATGTAGTGATTCTTAAGAGTGGGATAGAAATGATATTTTCATAAAATTTTATTTCGTTGTTCCCCAACTTGCATTGTCTGTAGAATTTCTTTTTGAAATTCTCTATGTTGGGGCCCCGTTCCCCAACTTGCACATTATTGTATGCTGACTTTTGTCAGCTTCTGTGTTGGGGCCCCGCCAACTTGCACATTATTGTAAGCTGACTTTCTGTCAGCTTCTATGTTGGGGCCCCGCCGATTTGTAAAAACATTAAAAACGATCATTTCTATTAAATCTGGCATAGATATGACCGTTTTTACTTTAATAAATAATATTGTATTTTAGGGAGTAAGACAGAAATATTAAAGAATCTCTAATGATTTAATATGTAGTGGTTCCTAAACATTAACCCAA
CRISPR-3的序列为
CCATACGTTAGAGATTCTTTATATTTCTGTCTTACTCCCTAAAATACAATATTATTTATTAAAGTAAAAACGGTCATATCTATGCCAGATTTAATAGAAATGATCGTTTTTAATGTTTTTACAAATCGGCGGGGCCCCAACATAGAAGCTGACAGAAAGTCAGCTTACAATAATGTGCAAGTTGGCGGGGCCCCAACACAGAAGCTGACAAAAGTCAGCATACAATAATGTGCAAGTTGGGGAACGGGGCCCCAACATAGAGAATTTCAAAAAGAAATTCTACAGACAATGCAAGTTGGGGAACAACGAAATAAAATTTTATGAAAATATCATTTCTATCCCACTCTTAAGAATCACTACATAATAAATCTTTAGTGGTTCTTTAACATTGATGTCACACTCCATGCCCATTGAGTAA
将测序得到的基因序列与Gene bank上基因序列进行BLAST比对，结果显示CRISPR-1与Staphylococcus aureus subsp. aureus NCTC 8325 chromosome 98%相同，CRISPR-2和CRISPR-3与Staphylococcus aureus subsp. aureus DSM 20231 93%相同。
（3）PCR扩增结果测序分析
对测序结果中3个CRISPR位点基因序列进行分析，结果显示3个CRISPR位点均有2个重复序列和一个间隔序列组成，如图4、图5、图6所示。
将3种CRISPR位点的间隔序列依次在Gene bank数据库进行BLAST比对，发现CRISPR-1的间隔序列与Staphylococcus aureus subsp. aureus NCTC 8325 chromosome完全匹配，CRISPR-2和CRISPR-3的间隔序列与Staphylococcus aureus subsp. aureusDSM 20231完全匹配，证实细菌样品CRISPR位点序列扩增成功。
本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。

Claims (7)

1.一种金黄色葡萄球菌CRISPR位点检测试剂盒，其特征在于，包括针对金黄色葡萄球菌的CRISPR位点基因序列设计的引物，所述金黄色葡萄球菌的CRISPR位点基因序列有三种，分别为CRISPR-1，CRISPR-2和CRISPR-3，对应的引物序列如下所示：
CRISPR-1 上游引物 5’-GTAGTGATTTTTGGGAGTGG-3’,
下游引物 5’-GTGGTTAATGTGTAGGGACC-3’,
CRISPR-2 上游引物 5’-CAATGACTTGGAGTGTGA-3’,
下游引物 5’-GGTTAATGTGTAGGAACC-3’,
CRISPR-3 上游引物 5’-ACTCAATGGCATGGAGTGTGA-3’,
下游引物 5’-GGTGGTTAATGTGTAGGGACC-3’。
2.根据权利要求1所述的一种金黄色葡萄球菌CRISPR位点检测试剂盒，其特征在于，还包括10×Tag Buffer，0.2 mM dNTP，2 mM MgCl2，0.05U/μL Tag DNA聚合酶，灭菌超纯水和DNA Marker 2000。
3.一种金黄色葡萄球菌CRISPR位点检测方法，其特征在于，包括以下步骤：
（1）以细菌样品的基因组DNA为模板，分别利用3种CRISPR位点基因序列的引物进行PCR扩增，引物序列如下所示，
CRISPR-1 上游引物 5’-GTAGTGATTTTTGGGAGTGG-3’,
下游引物 5’-GTGGTTAATGTGTAGGGACC-3’,
CRISPR-2 上游引物 5’-CAATGACTTGGAGTGTGA-3’,
下游引物 5’-GGTTAATGTGTAGGAACC-3’,
CRISPR-3 上游引物 5’-ACTCAATGGCATGGAGTGTGA-3’,
下游引物 5’-GGTGGTTAATGTGTAGGGACC-3’，
待PCR扩增结束后，得扩增产物；
（2）将所得扩增产物经凝胶电泳分析，CRISPR-1的预期扩增片段长度为330bp，CRISPR-2的预期扩增片段长度为380bp，CRISPR-3的预期扩增片段长度为387bp，得到相应大小的基因片段即为扩增成功。
4.根据权利要求3所述的一种金黄色葡萄球菌CRISPR位点检测方法，其特征在于，所述PCR扩增的反应体系为：10μmol/L上游引物1μL，10μmol/L下游引物1μL，10×Tag Buffer2.5μL，0.2 mM dNTP 0.5μL，2 mM MgCl2 2μl，0.05U/μL Taq DNA 聚合酶0.125μL，细菌样品基因组DNA 2μL，灭菌超纯水15.875μL。
5.根据权利要求3所述的一种金黄色葡萄球菌CRISPR位点检测方法，其特征在于，针对不同的CRISPR位点基因序列所采用的PCR扩增反应程序分别为：
CRISPR-1 94℃预变性5min；94℃变性30s，51℃退火30s，72℃延伸30s，共35个循环，72℃继续延伸10min；
CRISPR-2 94℃预变性5min；94℃变性30s，50℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min；
CRISPR-3 94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸45s，共35个循环，72℃继续延伸10min。
6.根据权利要求3所述的一种金黄色葡萄球菌CRISPR位点检测方法，其特征在于，凝胶电泳的条件为：1.5%琼脂糖凝胶，电压5V/cm，时间25min。
7.根据权利要求3所述的一种金黄色葡萄球菌CRISPR位点检测方法，其特征在于，细菌样品的基因组DNA采用煮沸法提取。
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