CN110241237B - 一种用于产气肠杆菌检测的试剂盒 - Google Patents
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Abstract
本发明提供一种用于产气肠杆菌检测的试剂盒,包含一种特异性靶向产气肠杆菌rpoB基因的向导RNA、扩增引物对、水化TwistAmp basic kit反应干燥球、LbCas12a蛋白、单链DNA探针(ssDNA)、Ribonuclease Inhibitor和缓冲液。使用该试剂盒进行产气肠杆菌检测,检测的特异性高,可以将产气肠杆菌与其他肠杆菌属细菌,包括阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌区分开来。同时,使用本发明所述RNA序列进行检测,耗时在1小时左右,不需要PCR仪,对设备要求低,显著低于传统的细菌培养法或PCR测序法,临床实用价值大。
Description
技术领域
本发明属于致病菌检测领域,涉及一种用于产气肠杆菌检测的试剂盒。利用此试剂盒可以进行产气肠杆菌快速检测。
背景技术
产气肠杆菌是人体感染的革兰阴性菌中常见菌群,给患者及社会带来巨大负担。传统的检测方法是进行细菌培养或PCR联合测序,但细菌培养需要5-7天的时间,而PCR常由于缺乏特异性引物,无法将产气肠杆菌与其他肠杆菌属细菌区分,故而需要联合一代测序才能鉴定,成本高,需要数天时间才能出结果。在这期间临床医师由于没有获得检测结果,只能凭经验用药,常常导致疾病治疗的延误。因而,如何快速检测产气肠杆菌显得尤为重要。近期,张峰等人发现,LbCas12a蛋白可用于致病菌的检测,只需要1小时左右即可完成检测,省时省力,灵敏度高。具体的原理为,该系统包含LbCas12a蛋白、致病菌DNA、向导RNA、两端分别带荧光基团和淬灭基团的单链DNA(ssDNA)。其中,致病菌DNA序列需要包含TTTN的PAM结构,同时该序列需要具备特异性,即区别于别的致病菌。而向导RNA序列为致病菌特异性靶向序列中TTTN后的一段序列,长度为20-24个碱基。在向导RNA引导下,LbCas12a蛋白可结合到致病菌特异性DNA序列上,随后,LbCas12a蛋白可表现出Dnase活性,从而切割两端分别带荧光基团和淬灭基团的单链DNA,使得荧光基团和淬灭基团分离,出现荧光,从而指示致病菌的存在。
发明内容
本发明的目的在于提供一种用于产气肠杆菌检测的试剂盒,包含一种特异性靶向产气肠杆菌rpoB基因的向导RNA、RPA扩增引物对、水化TwistAmp基础试剂盒(TwistAmpbasic kit)反应干燥球、LbCas12a蛋白、单链DNA探针(ssDNA)、RNA酶抑制剂(RibonucleaseInhibitor)和缓冲液。其中特异性靶向产气肠杆菌rpoB基因的向导RNA,其核苷酸序列如SEQ No.1所示,能特异性靶向产气肠杆菌,而不能靶向阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌,可用于产气肠杆菌的检测。RPA扩增引物对RPA-F和RPA-R,序列如SEQ No.2和SEQNo.3所示。单链DNA探针的序列如SEQ No.6所示。
本发明提供所述试剂盒进行产气肠杆菌快速检测的方法,通过以下步骤实现:
具体步骤为:取1ml样品,95-98摄氏度加热5min,取1μl作为检测样品,加入14.75μl水化TwistAmp basic kit反应干燥球(TwistDx公司),0.9μl 10mM RPA-F(序列为SEQNo.2)和RPA-R(序列为SEQ No.3),0.375μl Ribonuclease Inhibitor(Takara公司),3.5μlbuffer2.1(NEB公司),1000nM向导RNA(序列为SEQ No.1),250nM Cas12a(NEB公司),200nM单链DNA探针(上海生工,序列为SEQ No.6),加水补至25μl。37摄氏度反应25-45min,通过荧光显微镜下直接观察检测体系荧光变化,进行产气肠杆菌检测;发黄绿色荧光说明有产气肠杆菌污染,不发光说明没有产气肠杆菌污染。本方法可以将产气肠杆菌与其他肠杆菌属细菌,包括阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌区分开来。
其中:
所述单链DNA探针ssDNA序列(SEQ No.6)为:5’6-FAM-TTATT-3’BHQ1,由上海生工合成。
使用本发明所述检测试剂盒进行产气肠杆菌感染检测的优势在于:(1)与传统细菌培养或PCR联合一代测序法比较,传统方法需要数天时间,本方法只需要1小时即可完成检测。同时,本方法采用RPA法扩增,只要恒温水浴锅即可完成反应,不需要PCR仪,对设备要求低。(2)使用本发明所述检测试剂盒进行检测时,只有产气肠杆菌呈阳性反应,而其他种类的肠杆菌属细菌,包括常见的阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌均呈阴性反应。故而,本发明所述检测试剂盒具有高特异性。
附图说明
图1为产气肠杆菌、阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌rpoB基因序列比对图。
图2为样品荧光显微镜下检测结果,从1号-4号管样品加入的是各肠杆菌属细菌菌株,分别为产气肠杆菌、阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌。其中1号管有黄绿色荧光,呈阳性反应。2-4号管无黄绿色荧光,呈阴性反应。
图3为使用PCR法扩增不同种属肠杆菌,得到大小相似的条带,无法区分出产气肠杆菌。
图4为使用基于本发明所述向导RNA的检测法与传统PCR联合测序法,实验耗时比较图。
具体实施方式
下面结合附图和实施例对本发明作进一步的说明。
实施例1:肠杆菌属细菌rpoB基因序列比对
如图1所示,在产气肠杆菌rpoB基因序列中,本发明所述向导RNA序列(选中部分)的5’端为TTTN的PAM结构。该向导RNA序列在阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌中没有完全一致的序列,均有数个碱基的差异。且在阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌中的同源序列5’端均无TTTN的PAM结构,故而,使用本向导RNA,Cas12a蛋白无法靶向至阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌,因而,在检测时,只有产气肠杆菌可呈现阳性反应,其余肠杆菌属细菌呈阴性反应,可用于产气肠杆菌的特异性检测。
实施例2:本发明所述向导RNA特异性判定,步骤如下:
(1)准备产气肠杆菌、阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌标准菌株;
(2)取1ml待测样品,98摄氏度加热5min,吸取1μl作为检测样品;
(3)准备反应体系,反应体系为25μl,包括1μl检测样品,14.75μl水化TwistAmpbasic kit反应干燥球(TwistDx公司),0.9μl 10mM RPA-F(序列为SEQ No.2)和RPA-R(序列为SEQ No.3),0.375μl Ribonuclease Inhibitor(Takara公司),3.5μl buffer2.1(NEB公司),1000nM向导RNA(序列为SEQ No.1),250nM Cas12a(NEB公司),200nM单链DNA探针(上海生工),加水补至25μl;
(4)37摄氏度恒温反应30min;
(5)反应结束后,将PCR管置于荧光显微镜下直接观察,有荧光证明样品中存在产气肠杆菌,没有荧光则表示无产气肠杆菌污染。
检测结果如图2所示,样本1为产气肠杆菌,观察到荧光,呈阳性反应。其余样本非产气肠杆菌,无荧光,呈阴性反应。
实施例3:基于本发明所述向导RNA的检测法与传统PCR法特异性及实验耗时比较
1.基于本发明所述向导RNA的检测法,步骤如下:
(1)准备产气肠杆菌、阴沟肠杆菌、杰高维肠杆菌、阪崎肠杆菌标准菌株;
(2)取1ml待测样品,98摄氏度加热5min,吸取1μl作为检测样品;
(3)准备反应体系,反应体系为25μl,包括1μl检测样品,14.75μl水化TwistAmpbasic kit反应干燥球(TwistDx公司),0.9μl 10mM RPA-F(序列为SEQ No.2)和RPA-R(序列为SEQ No.3),0.375μl Ribonuclease Inhibitor(Takara公司),3.5μl buffer2.1(NEB公司),1000nM向导RNA(序列为SEQ No.1),250nM Cas12a(NEB公司),200nM单链DNA探针(上海生工),加水补至25μl;
(4)37摄氏度恒温反应30min;
(5)反应结束后,将PCR管置于荧光显微镜下直接观察,有荧光证明样品中存在产气肠杆菌,没有荧光则表示无产气肠杆菌污染;
(6)检测结果如图2所示,样本1为产气肠杆菌,观察到荧光,呈阳性反应。其余样本非产气肠杆菌,无荧光,呈阴性反应。
2.传统PCR法实验步骤如下:
(1)设计引物,PCR引物序列为RPA引物5’端起始的20个碱基,使获得的PCR产物与RPA产物序列相同,引物对序列见SEQ No.4和SEQ No.5;
(2)取1ml待测样品,98摄氏度加热5min,吸取1μl作为检测样品;
(3)配置反应体系,包含12.5μl DNA聚合酶预混液(上海生工),1μl正向引物,1μl反向引物,1μl待测样品,9.5μl水;
(4)PCR反应;
(5)琼脂糖凝胶电泳;
(6)实验结果如图3所示,所有的样本均扩增出232bp大小片段,无法区别产气肠杆菌与其他种类肠杆菌。
如上所述,PCR法无法区分肠杆菌种类,故需要进行测序比对,实验成本高,用时长,可能延误病情。
如图4所示,使用基于本发明所述检测试剂盒的检测方法特异性高,耗时短,仅为1小时左右,而使用PCR法,由于不同种肠杆菌属细菌序列高度同源,在一段序列中差异常常仅为数个碱基,设计的引物常常所有菌株都能扩增,所得产物大小接近,无法通过琼脂糖凝胶区分,故而需要进行测序,实验耗时在2天左右。本发明所述方法耗时显著低于PCR组。
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tgctcagcca gttgctccag ctgattttgt t 31
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Unknown)
<400> 4
agaaagacaa gcgtgcgctt g 21
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Unknown)
<400> 5
tgctcagcca gttgctccag ct 22
<210> 6
<211> 5
<212> DNA
<213> 人工序列(Unknown)
<400> 6
ttatt 5
Claims (1)
1.一种用于产气肠杆菌检测的试剂盒,其特征在于,由特异性靶向产气肠杆菌rpoB基因的向导RNA、扩增引物对、水化TwistAmp基础试剂盒反应干燥球、LbCas12a蛋白、单链DNA探针、RNA酶抑制剂和缓冲液组成;其中特异性靶向产气肠杆菌rpoB基因的向导RNA的核苷酸序列如SEQ No.1所示,RPA扩增引物对的序列如SEQ No.2和SEQ No.3所示,单链DNA探针的序列如SEQ No.6所示。
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