CN117144052A - 引物对、红色毛癣菌rpa试纸条试剂盒及检测方法 - Google Patents
引物对、红色毛癣菌rpa试纸条试剂盒及检测方法 Download PDFInfo
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Abstract
本发明公开了引物对,所述引物对的核苷酸序列如SEQ ID No.1和SEQ ID No.2所示,所述引物对用于检测红色毛癣菌。本发明还提供了红色毛癣菌RPA试纸条试剂盒及检测方法。本发明能够实现红色毛癣菌的快速检测。
Description
技术领域
本发明涉及红色毛癣菌检测相关技术领域。更具体地说,本发明涉及一种引物对、红色毛癣菌RPA试纸条试剂盒及检测方法
背景技术
红色毛癣菌是最常见的引起皮肤癣菌病的真菌。目前红色毛癣菌的检测仍然以传统的镜检和培养为主。只要用显微镜在取下的标本中找到菌丝和(或)孢子,诊断即成立。直接镜检检测操作简单,允许临床医生快速获得检测结果制定治疗方案。但是标本制备及检测者的专业水平对结果影响较大,在临床实践中,假阴性率达到15-30%。真菌培养可以明确致病菌及其活力,且可以根据形态学鉴定至种属,但耗时久,需要4周及以上。
近些年开始引入分子生物学技术用于甲真菌病的直接检测,包括PCR扩增联合凝胶电泳、PCR扩增联合酶切电泳、PCR-ELISA、PCR-sequencing和多重real-time PCR,其优势在于敏感性和特异性均较高,并且不易受用药影响,但受到仪器设备的限制。
重组酶聚合酶扩增(Recombinase polymerase amplification assay,RPA)是英国公司TwistDx Inc于2006年开发的核酸等温扩增技术。RPA检测包含三个关键酶:重组酶、单链DNA结合蛋白(single-stranded DNA-binding protein,SSB)和DNA聚合酶。重组酶与引物结合形成核酸蛋白复合体,该复合体在双链DNA中寻找定位同源序列后引发链交换反应形成D环结构,SSB稳定被置换的DNA单链,随后重组酶解离,聚合酶在引物3’-OH端添加碱基启动聚合反应。RPA在37~42℃条件下,实现对目标片段有效等温扩增,20min即可完成反应,且操作简便。RPA技术结合测流层析试纸条(lateral flow dipstick,LFD)相结合形成RPA-LFD检测体系,不需要电泳仪、荧光仪监测设备,可在5min内完成对扩增产物的快速可视化检测。目前国际上尚无基于RPA-LFD检测红色毛癣菌的报道。
发明内容
本发明的一个目的是提供一种引物对、红色毛癣菌RPA试纸条试剂盒及检测方法,能够实现红色毛癣菌的快速检测。
为了实现本发明的这些目的和其它优点,本发明提供了引物对,所述引物对的核苷酸序列如SEQ ID No.1和SEQ ID No.2所示,所述引物对用于检测红色毛癣菌。
本发明还提供了红色毛癣菌RPA试纸条试剂盒,包括所述的引物对。
进一步地,还包括:SEQ ID No.3所示的探针;侧流层析试纸条。
进一步地,所述探针5’端用FAM修饰,3’端用C3-Spacer修饰。
进一步地,所述侧流层析试纸条的结合垫中设置有胶体金包被的抗FAM抗体。
进一步地,所述侧流层析试纸条上具有检测线和质控线,所述检测线处设置有链霉亲和素,所述质控线处设置有抗FAM抗体。
本发明还提供了红色毛癣菌检测方法,利用所述的红色毛癣菌RPA试纸条试剂盒检测红色毛癣菌。
进一步地,包括:提取待测样品的DNA;利用所述引物对和所述探针进行RPA扩增反应;利用所述侧流层析试纸条检测扩增产物。
本发明至少包括以下有益效果:
本发明设计了SEQ ID No.1和SEQ ID No.2所示的引物对,成功将RPA和侧流层析试纸条结合,用于检测红色毛癣菌,检测操作简单,具有较好的敏感性和特异性,检测速度快,适用于即时诊断,有较好的应用前景。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为红色毛癣菌assay敏感性检测结果;
图2为红色毛癣菌assay特异性检测结果。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。
本申请的实施例提供了引物对,所述引物对的核苷酸序列如SEQ ID No.1和SEQID No.2所示,所述引物对用于检测红色毛癣菌;引物对的设计无标准流程,需要在满足一下规则的情况下,进行创造性劳动:1)引物长度一般在15-30碱基之间;2)引物GC含量在40%-60%之间,Tm值最好接近72℃;3)引物3′端要避开密码子的第3位;4)碱基要随机分布;5)引物自身及引物之间不应存在互补序列;6)引物应具有特异性。SEQ ID No.1:CTGTTCGAGCGTCATTTCAACCCCTCAAGC;SEQ ID No.2:5’-biotin-CCTGAAAAGATATATATATATATAAAGATTGGG;SEQ ID No.2引物的5’端由生物素biotin标记。
本申请的实施例还提供了红色毛癣菌RPA试纸条试剂盒,包括所述的引物对,利用引物对及RPA反应原料进行RPA反应,实现RPA扩增,随后利用试纸条进行检测。
在另一个实施例中,还包括:SEQ ID No.3所示的探针;侧流层析试纸条;优选地,序列SEQ ID No.3为:
5’-FAM-TCCGGCTTCCTAGGCGAATGGGCAGCCAATT/idSp/AGCGCCCTCAGGACCG-C3Spacer-3’,5’端用FAM修饰,3’端用C3-Spacer修饰,idsp表示碱基缺失;试纸条一般包括:样品垫、结合物释放垫、固定抗体的膜、吸附垫和加样孔,试纸条的各个部分固定在一个惰性的背衬材料上。
在另一个实施例中,所述侧流层析试纸条的结合垫中设置有胶体金包被的抗FAM抗体;试纸条的结合垫中预先固定有胶体金包被的兔来源的抗FAM抗体;胶体金中心为金原子凝聚成的金颗粒,周围吸附了一层负离子,使得胶体金带负电荷,可以在蛋白质/核酸上做标记,且不影响蛋白质/核酸的活性,同时喷涂有胶体金标记兔IgG,为胶体金和兔IgG结合体。
在另一个实施例中,所述侧流层析试纸条上具有检测线和质控线,所述检测线处设置有链霉亲和素,所述质控线处设置有抗FAM抗体;优选地,检测线和质控线利用硝酸纤维膜包被,距离加样孔近端为检测线,包被有能够识别生物素的链霉亲和素(streptavidin,SA),远端为质控线,包被有鼠抗兔IgG抗体;扩增产物加入加样孔后,样本在毛细作用下沿试纸条由近向远移动,样本中5’端为FAM标记的产物将在结合垫处与胶体金包被的抗FAM抗体结合形成复合物;层析至检测线处(T线),T线固定的链霉亲和素与3’端为生物素标记的扩增产物结合,胶体金在此处发生聚集沉淀而显示红色,这是由于聚集而颗粒变大的光学效应;未被捕获的胶体金包被的兔来源的抗FAM抗体层析至质控线处(C线),C线固定的抗兔IgG抗体与之结合,胶体金在此聚集沉淀而显示红色,证明层析过程顺利完成,检测试纸条工作正常。
本申请的实施例还提供了红色毛癣菌检测方法,利用所述的红色毛癣菌RPA试纸条试剂盒检测红色毛癣菌,即利用引物对、探针、试纸条等材料对红色毛癣菌进行检测。
在另一个实施例中,包括:S1:提取待测样品的DNA,可选地,采用有机溶剂提取法或Chelex-100提取法;优选地,采用chelex-100加热煮沸法提取DNA;Chelex-100是一种由苯乙烯、二乙烯苯共聚体组成的化学螯合树脂,在低离子强度、碱性及煮沸的条件下,可以使细胞膜破裂,并使蛋白质变性,通过离心除去Chelex颗粒,使其结合的物质与DNA分离。
S2:利用所述引物对和所述探针进行RPA扩增反应;可选地,将提取的DNA、引物对、探针、缓冲液、模板、无核酸酶水混合进行RPA反应
S3:利用所述侧流层析试纸条检测扩增产物;同时出现T线和C线为阳性(+),仅出现C线为阴性(-),只出现T线需考虑试纸条是否有效。
以下以具体实施例说明:
样本处理:利用chelex-100加热煮沸法提取待测样品DNA;
5% Chelex-100裂解液的配制,称量5g Chelex-100、吸取1mL的TritonX-100溶于100mL 1×TE缓冲液后,混匀后标记备用。收集待测样品加入1.5mL EP管中,放入适量玻璃珠以及200μL的5% Chelex-100裂解液,震荡10min,置于金属浴中100℃加热10min。加热煮沸的粗提取液12 000rpm/min离心5min,将上清液转移到新的1.5mL,即为待测样品DNA提取产物。
反应体系如表1所示:
表1反应体系试剂用量
反应条件:将表1中共47μL的RPA反应体系混合;手弹使冻干粉(正向引物、反向引物和探针可使用冻干粉形式)充分溶解,并短暂离心;反应盖上加3ul启动剂,盖紧管盖,短暂离心使启动剂加入预混液中,手弹混匀短暂离心;将反应管放入相应恒温加热仪(39℃)20分钟;取96孔板,每孔加入100ul缓冲液(Milenia Hybridtech核酸扩增快速检测试纸条内配备的缓冲液),加入10ul反应产物充分混匀后,加入试纸条的加样孔内,观察结果。
结果判读:同时出现T线和C线为阳性(+),仅出现C线为阴性(-),只出现T线需考虑试纸条是否有效。
敏感性及特异性检测:
选取一株红色毛癣菌价其检测效力,利用DNeasy Plant Mini Kit植物DNA提取试剂盒提取真菌基因组DNA。
提取所得真菌基因组DNA经NanoDrop 2000微量紫外分光光度计准确定量,用灭菌注射用水准确稀释至0.5×10ng/μL后,10倍稀释得到0.5×10ng/μL-0.5×100fg/μL 6个系列稀释标准品,以无菌水作为NTC模板。将0.5×10ng/μL-0.5×100fg/μL的基因组DNA标准品按照上述方法,每个反应体系加入2μL模板(真菌基因组DNA),即每孔10ng-×100fg基因组DNA标准品按照上述条件在39℃恒温加热仪加热扩增后用测流层析试纸条进行检测,将试纸条拍照记录。结果如图1所示,根据检测结果可知,本申请的检测下限是能够稳定扩增的最低模板量为10pg/反应,具有较好的敏感性。
提取红色毛癣菌、须癣毛癣菌、絮状表皮癣菌、犬小孢子菌、断发毛癣菌、趾间毛癣菌、白色念珠菌、近平滑念珠菌、糠秕马拉色菌、球型马拉色菌、镰刀菌、青霉菌、土曲霉、黄曲霉、烟曲霉、枝顶孢、孢子丝菌、疣状瓶霉等的DNA,进行特异性检测,结果如图2显示,本申请的引物对、探针特异性能够扩增红色毛癣菌,对另外17种非红色毛癣菌真菌无交叉反应。
这里说明的设备数量和处理规模是用来简化本发明的说明的。对本发明引物对、红色毛癣菌RPA侧流层析试纸条试剂盒及检测方法的应用、修改和变化对本领域的技术人员来说是显而易见的。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
Claims (8)
1.引物对,其特征在于,所述引物对的核苷酸序列如SEQ ID No.1和SEQ ID No.2所示,所述引物对用于检测红色毛癣菌。
2.红色毛癣菌RPA试纸条试剂盒,其特征在于,包括权利要求1所述的引物对。
3.如权利要求2所述的红色毛癣菌RPA侧流层析试纸条试剂盒,其特征在于,还包括:
SEQ ID No.3所示的探针;
侧流层析试纸条。
4.如权利要求3所述的红色毛癣菌RPA试纸条试剂盒,其特征在于,所述探针5’端用FAM修饰,3’端用C3-Spacer修饰。
5.如权利要求4所述的红色毛癣菌RPA试纸条试剂盒,其特征在于,所述侧流层析试纸条的结合垫中设置有胶体金包被的抗FAM抗体。
6.如权利要求5所述的红色毛癣菌RPA试纸条试剂盒,其特征在于,所述侧流层析试纸条上具有检测线和质控线,所述检测线处设置有链霉亲和素,所述质控线处设置有抗FAM抗体。
7.红色毛癣菌检测方法,其特征在于,利用如权利要求6所述的红色毛癣菌RPA试纸条试剂盒检测红色毛癣菌。
8.如权利要求7所述的红色毛癣菌检测方法,其特征在于,包括:
提取待测样品的DNA;
利用所述引物对和所述探针进行RPA扩增反应;
利用所述试纸条检测扩增产物。
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