CN116179727A - 一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒 - Google Patents
一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒,引物组由外引物F3基因序列、外引物B3基因序列、内引物FIP基因序列、内引物BIP基因序列组成;试剂盒包括环介导等温扩增反应液、显色剂、反应酶液、嗜肺军团菌阳性质控标准品和假阳性对照的无核糖核酸的去离子水。试剂盒检测的特异性强,灵敏度高,检测速率快,仅需45分钟就可得到结果;实用性高,检测成本低,不需要昂贵的精密仪器。
Description
技术领域
本发明是关于生物检测的技术领域,特别是关于一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒。
背景技术
肺炎是一种高度致命性的呼吸道疾病,其感染病原菌主要是革兰阴性杆菌。军团菌是一种革兰氏兼性细胞内致病菌,广泛存在于自然和人工水系中,嗜肺军团菌是引起人类军团菌病的主要病原菌,可引起80%-90%军团菌病的暴发流行。
军团菌病的初期表现和流感类似,因此初期通过临床表现很难区分病原,只有通过实验室的遗传物质诊断才能鉴别。
鉴别病原菌的方法包括病毒离体培养、酶联免疫法、聚合酶链式反应法(Polymerase Chain Reaction,PCR)等。病毒分离培养法试验周期长,操作复杂,不能实时监测。酶联免疫法受检测仪器和环境影响较大,易出现假阳性结果。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明的目的在于提供一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒,其能够。
为实现上述目的,本发明提供了一种嗜肺军团菌的环介导等温扩增检测引物组,由外引物F3基因序列、外引物B3基因序列、内引物FIP基因序列和内引物BIP基因序列组成,具体序列如下:
F3:AGTGGTTTGCAATACAAAGT(SEQ ID No.1);
B3:CCATATGCAAGACCTGAGG(SEQ ID No.2);
FIP:ACGGTACCATCAATCAGACGACGTTAAACCCGGAAAATCGGA(SEQ ID No.3);
BIP:
CAACGTTCCAGGTTTCACAAGTTATAAATTTCCCAAGTTGATCCA(SEQ ID No.4)。
本发明还提供一种嗜肺军团菌的环介导等温扩增检测试剂盒,包括上述的嗜肺军团菌的环介导等温扩增检测引物组。
进一步地,还包括环介导等温扩增反应液、显色剂和反应酶液。
进一步地,还包括嗜肺军团菌阳性质控标准品和假阳性对照。
优选地,所述假阳性对照为无核糖核酸的去离子水。
优选地,所述环介导等温扩增反应液包括:Betain、dNTPs、10×Buffer和浓度为100mM的MgSO4。
优选地,所述显色剂为SYBR Green I荧光显色剂或钙黄绿素显色剂。
优选地,所述反应酶液为Bst DNA聚合酶。
优选地,所述试剂盒内环介导等温扩增检测的反应体系包括4μL Betain(5M),2.5μL dNTPs(10mM each),2.5μL 10×Buffer(由200mM Tris-HCl、100mM(NH4)2SO4、500mMKCl、1% Tween20组成),1.5μL MgSO4(100mM),引物F3和B3各0.1μL,引物FIP和BIP各0.25μL,1μL BST DNA聚合酶,1μL样品DNA,ddH2O补足至25μL。
与现有技术相比,根据本发明的一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒,具有以下优点:
1、针对嗜肺军团菌基因核糖体内转录区间序列,设计4个特异性引物,设计的引物数量小,并且能够使扩增产物具有高特异性;
2、环介导等温扩增检测反应在恒温条件下进行,仅需要一台恒温加热装置,降低检测成本,检测流程简单,观测荧光发射光谱,可直观反应是否有病原菌以及菌数量,检测速度快,反应时间仅40-50min;
3、试剂盒的检出率高,准确性高,检出范围大,灵敏度高。
附图说明
图1是根据本实施例2的电泳结果示意图。
具体实施方式
下面结合附图,对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
除非另有其它明确表示,否则在整个说明书和权利要求书中,术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分,而并未排除其它元件或其它组成部分。
根据本发明优选实施方式的一种嗜肺军团菌的环介导等温扩增检测引物组及试剂盒,针对嗜肺军团菌基因核糖体内转录区间序列(SEQ ID No.5),设计4个嗜肺军团菌具有特异性扩增作用的环介导等温扩增引物,使扩增产物具有高特异性;采用环介导等温扩增技术进行检测,反应在恒温条件下进行,仅需要一台恒温加热装置,降低检测成本。
核糖体内转录区间序列(SEQ ID No.5)的LAMP引物设计思路如下:
TGTAGAAGGATATTACCTTTTTGTCCATTATATAATTAAATGATAGCTTATGACTGGTAATTTTACGCAAATTTAGGCAGAATTAGTGGGCGATTTGTTTTTGTTTAATTTTGCTCAATTTATTGTGCAGTATGAGAACTTAAGTGTAAGACTAAAAGGGGATTGTTTATGAAGATGAAATTGGTGACTGCAGCTGTTATGGGGCTTGCAATGTCAACAGCAATGGCTGCAACCGATGCCACATCATTAGCTACAGACAAGGATAAGTTGTCTTATAGCATTGGTGCCGATTTGGGGAAGAATTTTAAAAATCAAGGCATAGATGTTAATCCGGAAGCAATGGCTAAAGGCATGCAAGACGCTATGAGTGGCGCTCAATTGGCTTTAACCGAACAGCAAATGAAAGACGTTCTTAACAAGTTTCAGAAAGATTTGATGGCAAAGCGTACTGCTGAATTCAATAAGAAAGCGGATGAAAATAAAGTAAAAGGGGAAGCCTTTTTAACTGAAAACAAAAACAAGCCAGGCGTTGTTGTATTGCCAAGTGGTTTGCAATACAAAGT(F3)AATCAATTCTGGAAATGGTGTTAAACCCGGAAA ATCGGA(F2)TACAGTCACTGTCGAATATACTGGTCGTCTGATTGATGGTACCGT(F1)TTTTGACAGTACCGAAAAAACTGGTAAGCCAGCAACGTTCCAGGTTTCACAAGTT(B1)ATTCCTGGATGGACAGAAGCTTTGCAATTGATGCCCGCTGGATCAACTTGGGAAATTTAT(B2)GTTC CCTCAGGTCTTGCATATGG(B3)CCCACGTAGCGTTGGCGGACCTATTGGCCCAAATGAAACTTTAATATTTAAAATTCACTTAATTTCAGTGAAAAAATCATCTTAAGTTTTTTTGAATTAAAGTCATACAAAACGCATCCTTCTCATTTAGAGAGGGATGCTCTCTTTGTAAAGGCTAATGATCTTCATAACAGGTGTCAGCCTAACACCACTGAGGAAATTAAAATATGTCTGTTTTAAAGGCTATGACTCATACTGGTGAACAAAGGAATGAACAAGCGCCTGTTTATCGTTTTCATTTTAGGTTTTTCTTCCGGTTTACCTATGGCATTAATCAGCAGTACCTTGCAAGCATGGTACGCTAATAATGGCATGTCAGTGCTTGCGACGGGAGCTTTAAGTTTAGTCAGTCTACCTTATGCGTATCGTATTTTCTGGGGTCCAATTCTTGATCGCTACTCTTTATTTCATTTAGGGAAAAGGCGAAGCTGGATTCTCACCATGCAGATACTACTGCTTCTGGGTTTTAATCTCATGGCCTGGTTTATTCCAGCTCAATATCCTAAATTGATGGCATTTTTAGCTTTAGTATTAGCCTGTTTTTCAGCCACACAAGATGTGGCAATTGATGCTCATCGAGCAGAATATCTTCCCCCACAGGAACATGCATTGGGCGCGTCTCTGGCTGTGTTTGGGTATCGATTAGCTCTTTTGTTATCTGGCGGTTTGGCTTTGGTAATGGCAGAA。
环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)具有操作简单、高灵敏度、特异性强等优点。环介导等温扩增技术的原理是针对靶基因上的六个区域设计四条引物,利用链置换型DNA聚合酶在恒温条件下进行扩增反应,可在15-60分钟内实现大量扩增。与常规的聚合酶链式反应法相比,LAMP技术不需要模板热变性、温度循环等复杂的过程,在灵敏度、检测范围、特异性等指标检测方面优于PCR技术,可通过肉眼观察判断是否有待测基因的存在,是一种适合现场快速检测的方法。
嗜肺军团菌的环介导等温扩增检测引物组由外引物F3基因序列、外引物B3基因序列、内引物FIP基因序列、内引物BIP基因序列组成,具体序列如下:
F3:AGTGGTTTGCAATACAAAGT(SEQ ID No.1);
B3:CCATATGCAAGACCTGAGG(SEQ ID No.2);
FIP:ACGGTACCATCAATCAGACGACGTTAAACCCGGAAAATCGGA(SEQ ID No.3);
BIP:
CAACGTTCCAGGTTTCACAAGTTATAAATTTCCCAAGTTGATCCA(SEQ ID No.4)。
嗜肺军团菌的环介导等温扩增检测试剂盒,包括环介导等温扩增反应液、显色剂和反应酶液,还包括嗜肺军团菌阳性质控标准品和假阳性对照的无核糖核酸的去离子水。
环介导等温扩增反应液包括:4μL Betain,2.5μL dNTPs,2.5μL 10×Buffer,1.5μL MgSO4(100mM)。显色剂为SYBR Green I荧光显色剂或钙黄绿素显色剂。反应酶液为BstDNA聚合酶。
嗜肺军团菌的环介导等温扩增检测试剂盒内,LAMP反应体系包括4μLBetain(5M),2.5μL dNTPs(10mM each),2.5μL 10×Buffer(由200mM Tris-HCl、100mM(NH4)2SO4、500mMKCl、1% Tween20组成),1.5μL MgSO4(100mM),引物F3和B3各0.1μL,引物FIP和BIP各0.25μL,1μL BST DNA聚合酶,1μL样品DNA,ddH2O补足至25μL。
嗜肺军团菌的环介导等温扩增检测方法,具体包括以下步骤:
S1,提取样品DNA;
S2,将样本DNA与引物组、环介导等温扩增反应液、显色剂和反应酶液混合得到混合液;
S3,将混合液置于65℃恒温水浴中反应40-50min。
S4,反应中记录荧光发射光谱,判断样本里是否具有嗜肺军团菌,具有多少数量。
实施例1嗜肺军团菌的环介导等温扩增检测方法的建立
1.待测样品DNA提取:
采用天根生化科技(北京)有限公司微量样品基因组DNA提取试剂盒(DP316)提取样本DNA。取100μL样本储备液到1.5ml的离心管中,加入10μLProteinase K溶液。加入100μL的缓冲液GB,轻轻颠倒混匀,简短离心以去除管盖内壁的液滴。56℃温浴10min,并不时轻摇样品。加入50μL乙醇(体积分数为96-100%),如果室温超过25℃,请将乙醇置冰上预冷。轻轻颠倒混匀样品,室温放置3min。简短离心以去除管盖内壁的液滴。将上一步所得溶液都加到一个吸附柱CR2中,12000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。向吸附柱CR2中加入500μL缓冲液GD,12000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。向吸附柱CR2中加入600μL漂洗液PW,12000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。再向吸附柱CR2中加入600μL漂洗液PW,12000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。12000rpm离心2min,倒掉废液。将吸附柱CR2置于室温放置2-5min,以彻底晾干吸附材料中残余的漂洗液。将吸附柱CR2转入一个干净的离心管中,向吸附膜中间位置悬空滴加20-50μL洗脱缓冲液TB,室温放置2-5min,12000rpm离心2min,将溶液收集到离心管中。
2.构建LAMP扩增体系:
组分及反应条件如下:4μL Betain(5M),2.5μL dNTPs(10mM each),2.5μL 10×Buffer(由200mM Tris-HCl、100mM(NH4)2SO4、500mM KCl、1%Tween20组成),1.5μL MgSO4(100mM),引物F3和B3各0.1μL,引物FIP和BIP各0.25μL,1μL BST DNA聚合酶,1μL样品DNA,ddH2O补足至25μL。将上述所有试剂置于离心管中,65℃水浴45min进行扩增反应。
3.扩增结果分析:
反应在65℃下进行,每1min采集一次荧光信号,绘制荧光发射曲线。若曲线呈S型则样品中有嗜肺军团菌,否则没有。将荧光发射曲线和标准曲线进行比对,则可以定量测量样品中嗜肺军团菌的含量。
实施例2嗜肺军团菌的环介导等温扩增检测的特异性、准确性验证
采用本发明的试剂盒分别对嗜肺军团菌和非嗜肺军团菌进行检测,通过琼脂糖凝胶电泳对结果进行验证。
凝胶电泳检测方法:取2μL扩增产物,加入1μL上样缓冲液(购自上海康朗生物科技有限公司),混匀,在2%(W/V)琼脂糖凝胶中、10V/cm下电泳20min,用溴化乙锭染色后,在成像系统上显像,观察是否有梯形条带。
检测结果:结果如图1所示,嗜肺军团菌组经过扩增之后,琼脂糖凝胶电泳结果出现阶梯状条带,其中1-4泳道分别为10^5,10^4,10^3,10^2个嗜肺军团菌提取的DNA作为模板进行扩增的结果,5、6泳道为大肠杆菌和金黄色葡萄球菌提取的DNA用嗜肺军团菌试剂扩增的结果,说明本发明有较好的特异性和准确性。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
Claims (9)
1.一种嗜肺军团菌的环介导等温扩增检测引物组,其特征在于,由外引物F3基因序列、外引物B3基因序列、内引物FIP基因序列和内引物BIP基因序列组成,具体序列如下:
F3:AGTGGTTTGCAATACAAAGT(SEQ ID No.1);
B3:CCATATGCAAGACCTGAGG(SEQ ID No.2);
FIP:ACGGTACCATCAATCAGACGACGTTAAACCCGGAAAATCGGA(SEQ ID No.3);
BIP:
CAACGTTCCAGGTTTCACAAGTTATAAATTTCCCAAGTTGATCCA(SEQ ID No.4)。
2.一种嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,包括权利要求1所述的嗜肺军团菌的环介导等温扩增检测引物组。
3.如权利要求2所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,还包括环介导等温扩增反应液、显色剂和反应酶液。
4.如权利要求3所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,还包括嗜肺军团菌阳性质控标准品和假阳性对照。
5.如权利要求4所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,所述假阳性对照为无核糖核酸的去离子水。
6.如权利要求3所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,所述环介导等温扩增反应液包括:Betain、dNTPs、10×Buffer和MgSO4。
7.如权利要求3所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,所述显色剂为SYBR Green I荧光显色剂或钙黄绿素显色剂。
8.如权利要求3所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,所述反应酶液为Bst DNA聚合酶。
9.如权利要求3所述的嗜肺军团菌的环介导等温扩增检测试剂盒,其特征在于,所述试剂盒内环介导等温扩增检测的反应体系包括4μL Betain(5M),2.5μL dNTPs(10mM each),2.5μL 10×Buffer(由200mM Tris-HCl、100mM(NH4)2SO4、500mM KCl、1%Tween20组成),1.5μL MgSO4(100mM),引物F3和B3各0.1μL,引物FIP和BIP各0.25μL,1μL BST DNA聚合酶,1μL样品DNA,ddH2O补足至25μL。
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