CN114561393B - 一种特异性识别鸟-胞内分枝杆菌复合群的dna适配子及其筛选方法和应用 - Google Patents
一种特异性识别鸟-胞内分枝杆菌复合群的dna适配子及其筛选方法和应用 Download PDFInfo
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Abstract
本发明提供了一种特异性识别鸟‑胞内分枝杆菌复合群的DNA适配子及其筛选方法和应用。该DNA适配子的序列为SEQ ID NO.1。本发明在全细胞SELEX技术基础上利用构建进化发育树的方法系统地监测了整个筛选过程,进而获得了识别鸟‑胞内分枝杆菌复合群的DNA适配子MAC‑A11,其特异性高、亲和性好,可高特异性地识别鸟‑胞内分枝杆菌复合群,为MAC肺病的诊断和治疗提供了有力依据。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及一种特异性识别鸟-胞内分枝杆菌复合群(MAC)的DNA适配子及其筛选方法和应用。
背景技术
非结核分枝杆菌(NTM)是导致进行性肺部疾病的普遍存在的生物。近年来,世界许多地方,非结核分枝杆菌肺病的发病率逐年上升。在我国,所有分枝杆菌分离株中,NTM的分离率从1990年的4.9%上升到2010年的22.9%,呈明显上升趋势,已成为威胁人类健康的重要公共卫生问题之一。鸟-胞内分枝杆菌复合群(Mycobacteriumaviumcomplex,MAC)主要包括鸟分枝杆菌和胞内分枝杆菌,广泛存在于自然环境中,成为最常见的NTM。MAC感染可以引起人畜共患性传染病,可以侵害多种组织器官,包括肺、骨髓、淋巴结、皮肤软组织和播散性病变,以MAC肺病最为常见。MAC肺病与结核分枝杆菌感染引起的肺结核在临床表现、影像学特点及病理学组织极其相似,很多MAC肺病患者容易被漏诊或误诊为多耐药肺结核。MAC肺病对一线、二线抗结核药物呈高度耐药,若按肺结核方案治疗不仅疗效差、延误治疗时机,且药物不良反应风险高。因此,急需提高对MAC肺病和肺结核的早期鉴别诊断。
目前MAC肺病的实验室诊断主要依赖于分枝杆菌菌种的鉴定,主要检测技术包括生理生化反应、DNA探针法、PCR限制性片段长度多态性分析法、DNA测序法等。经典的细菌学生理生化反应检测方法时间长特异性低,无法满足临床快速诊疗需求。分子生物学诊断虽然能满足速度快、特异性高等要求,但其高成本,并且操作复杂,对实验条件和技术人员要求高,限制了其在临床检验中的普及应用。
指数富集的配体系统进化技术(Systematic evolution of ligand byexponential enrichment,SELEX)是Ellington与Szostak(1990)最先报道使用,是一种新型体外筛选技术。该技术的基本原理是利用寡核苷酸分子可在空间形成多种多样的三维结构,通过构建的随机寡核苷酸文库,从中筛选出与目标靶分子有特异识别作用的高亲和力的寡核苷酸分子,再经扩增、反复筛选,使该类寡核苷酸分子得到富集,该富集的寡核苷酸分子称为适配子。与抗体蛋白相比,适配子具有分子识别能力强、稳定性高、制备简单、经济、快速等优点。该技术已成功运用于许多靶分子的筛选,包括金属离子、有机染料、蛋白质、细胞、药物、氨基酸以及各种细胞因子等,可用于相应靶分子的检测识别。目前该技术在在人类病原微生物检测方面得到了广泛的应用。特别是对一些未知的致病性细菌或病毒的研究,虽然不知道其内部结构、功能以及这些物质的表位,但将其作为靶物质,通过SELEX过程筛选到与其相对应的适配子,检测靶物质,已成为该领域的研究探索热点。但目前未有采用SELEX技术获得与MAC相对应的适配子的报道。
发明内容
为了克服现有技术中的缺陷,本发明提供了一种特异性识别鸟-胞内分枝杆菌复合群的DNA适配子及其筛选方法和应用。
为实现上述目的,本发明采用如下技术方案:
本发明的第一方面是提供一种特异性识别鸟-胞内分枝杆菌复合群的DNA适配子,其序列为SEQ ID NO.1。
本发明的第二方面是提供上述适配子的筛选方法,包括如下步骤:
步骤一,对ssDNA文库采用PCR扩增和不对称PCR扩增进行纯化;
步骤二,利用SELEX技术筛选识别鸟-胞内分枝杆菌复合群的DNA适配子
(1)反筛:将反筛菌体(非分枝杆菌)与纯化后的ssDNA文库共同孵育,然后离心,弃掉沉淀,留取上清液;
(2)结合并回收:将上清液与靶标菌体共同孵育,然后离心,弃掉上清液;重悬沉淀以此为模板进行PCR扩增;回收产物进行下一轮筛选。
进一步地,在每一轮筛选之后的ssDNA文库中随机挑取一定数量的克隆测序,根据序列同源性构建进化发育树系统监测筛选过程,以了解这些适配子对目标细菌复杂结构的动态进化,提高菌体适配子的筛选成功率。
进一步地,上述筛选方法还包括采用ELISA方法测试适配子与靶菌体的亲和性。
进一步地,将生物素标记的ssDNA适配子与靶菌体共同孵育,然后加入稀释后的链酶亲和素标记的辣根过氧化物酶共同孵育;孵育完成,洗涤,加入显色液进行显色反应,最后采用酶标仪于450nm处测定OD值。
本发明的第三方面是提供上述DNA适配子在制备识别鸟-胞内分枝杆菌复合群试剂盒中的应用,该试剂盒包括该DNA适配子。
进一步地,该DNA适配子采用生物素、荧光素或酶标记。
本发明的第四方面是提供上述DNA适配子在制备MAC肺病诊断试剂盒中的应用,该试剂盒包括该DNA适配子。
进一步地,该DNA适配子采用生物素、荧光素或酶标记。
本发明的第五方面是提供一种鸟-胞内分枝杆菌复合群的三明治夹心ELISA检测方法,采用上述DNA适配子作为捕获适配子,5'端生物素标记的另一个适配子作为检测适配子。
本发明的第六方面是提供一种检测鸟-胞内分枝杆菌复合群的方法,用荧光标记上述DNA适配子,预处理之后和待测菌体温和振荡30-60min,离心,重悬沉淀,置于荧光显微镜下观察。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
本发明在全细胞SELEX技术基础上利用构建进化发育树的方法系统地监测了整个筛选过程,进而获得了识别鸟-胞内分枝杆菌复合群的DNA适配子MAC-A11,其特异性高、亲和性好,可高特异性地识别鸟-胞内分枝杆菌复合群,为MAC肺病的诊断和治疗提供了有力依据。
附图说明
图1是MAC-A11适配子的二级结构示意图;
图2是本发明一实施例中MAC组、结核分枝杆菌(MTB)组和非分枝杆菌组在检测波长为450nm下的吸光度;
图3是本发明一实施例中临床菌株(鸟分枝杆菌标准株ATCC 25291、胞内分枝杆菌标准株ATCC 13950、标准株H37R、无毒株H37Ra、大肠杆菌和金黄色葡萄球菌)与适配子MAC-A11的荧光显微镜结果图。
具体实施方式
本发明提供了一种特异性识别鸟-胞内分枝杆菌复合群的DNA适配子及其筛选方法和应用。为了获得高度特异性的MAC适配子作为诊断工具,在SELEX筛选过程中,本发明人设置了多种对照。利用空白对照预选,在前三轮中消除了具有混杂结合特性的适配子。在中间四轮或最后五轮期间,通过利用非分枝杆菌和结核菌标准株H37Rv对照预选来消除与其他病原菌和结核菌结合的适配子。通过12轮筛选,成功获得了可高特异性识别胞内分枝杆菌的适配子。此外,利用构建进化发育树的方法系统地监测了整个筛选过程,以了解这些适配子对目标细菌复杂结构的动态进化,提高了对菌体适配子的筛选成功率。
上述DNA适配子为MAC-A11,其核苷酸序列为GGGAGCTCAGAATAAACGCTCAACCGGATCGCGCGATAGACTAGACGTGTTACGTTTGTTCGACATGAGGCCCGGATC(SEQ ID NO.1)。
下面通过具体实施例和附图对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
实施例中方法如无特殊说明的采用常规方法,使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1
本实施例通过SELEX技术筛选与MAC相对应的适配子,具体的筛选过程和结果如下:
1.筛选菌株的制备
将胞内分枝杆菌标准株ATCC 13950、结核分枝杆菌标准株H37Rv转种至含有10%OADC(含油酸、白蛋白、葡萄糖和过氧化氢酶)营养添加剂的米氏7H9液体培养基中,37℃培养至对数生长期,转至1.5ml离心管中,12000rpm离心5min,1×PBS洗涤两遍,80℃水浴,灭火30min。灭火后转至磨菌管,磨菌后调整浊度至1mg/ml,备用。
非分枝杆菌均从血平板上刮取生长良好的菌落,用上述同样的方法处理灭火备用。
2.PCR扩增反应
2.1ssDNA文库PCR扩增得dsDNA
按照表1配置PCR反应体系,以ssDNA文库(参见申请号为CN201510379009.2的专利)为模板,PCR反应热力学循环参数为:95℃预变性5min;94℃预变性30s,65℃退火30s,72℃延伸30s,反应18个循环;72℃延伸5min,4℃保存。
2.2PCR产物电泳及回收
配制2%的琼脂糖凝胶,精确称取2g电泳级琼脂糖,加入100ml 1×TAE电泳缓冲液,在微波炉内加热至融化,待凝胶冷却至55℃左右时,加入溴化乙锭(终浓度为0.5μg/ml)轻轻旋转混匀,倒入胶模,插入合适的梳子,让凝胶溶液凝结后使用。
表1 PCR反应体系组成
(1)PCR产物电泳:
将凝胶置于电泳槽,取5μl PCR产物加入1μl 6×loading buffer混合后上样,另取5μl DL2000 Marker,分别加入琼脂糖凝胶孔内,在1×TAE电泳缓冲液中设置程序为100V、20min,在凝胶成像仪上观察电泳结果。
(2)PCR产物回收:
往盛有待纯化的DNA溶液的1.5ml EP管中加入0.1倍体积的3mol/L pH5.2的乙酸钠(NaAc)和2~2.5倍体积的无水乙醇,在蜗旋混合振荡器上振荡混匀并置于-20℃ 30min,然后12000rpm离心10min,弃上清,加入1ml预冷的70%乙醇,颠倒离心管数次,12000rpm离心10min,弃上清,晾干沉淀后溶于20μlddH2O。
2.3不对称PCR扩增
按照表2配置PCR反应体系,以dsDNA文库为模板,PCR反应热力学循环参数为:95℃预变性5min;94℃预变性30s,65℃退火30s,72℃延伸30s,反应40个循环;72℃延伸5min,4℃保存。
表2不对称PCR反应体系组成
3.SELEX筛选
以1.5ml的EP管作为筛选介质的SELEX筛选是主要基于离心沉淀法。
(1)准备ssDNA文库:将用1×PBS(pH 7.4)缓冲液稀释好的单链寡核苷酸文库94℃变性5min,并室温冷却15min。取单链寡核苷酸文库200μl加入新的1.5ml EP管中37℃孵育45min,12000rpm离心5min,取上清,移至新EP管中,目的是去除与EP管管壁结合的ssDNA。
(2)反筛:取适量的反筛菌体(非结核分枝杆菌)加入至含有核酸文库的EP管中,37℃孵育45min,12000rpm离心5min,弃沉淀,将上清移至新EP管中,去除与反筛菌体特异性结合的ssDNA。
(3)结合并回收:再加入适量的靶标菌体37℃孵育45min,12000rpm离心5min,弃上清,除去未跟靶标菌体结合的ssDNA,留下ssDNA-菌体结合物的沉淀。用20μl ddH2O重悬沉淀,以此作为模板,进行PCR扩增,回收产物,准备下一轮的SELEX筛选。SELEX筛选参数可详见表3。每轮筛选后的ssDNA文库克隆后,随机挑取一定数量的克隆测序,根据序列同源性构建进化发育树系统监测筛选过程,以了解这些适配子对目标细菌复杂结构的动态进化,提高菌体适配子的筛选成功率。筛选后的饱和文库,经克隆测序,获得单个适配子并进行二级结构分析和亲和性检测。
表3 SELEX筛选中各轮孵育时间,菌体和ssDNA的反应浓度
注:非分枝杆菌:嗜麦芽寡养单胞菌、粘质沙雷氏菌、弗氏柠檬酸杆菌、肺炎克雷伯菌和大肠杆菌。
3.亲和性检测
为了确定适配子的亲和性,用0.1mmol/L的碳酸盐缓冲液(pH9.6)将靶标菌体(1×105CFU/孔)包被于96微孔聚苯乙烯酶标板中,4℃过夜。次日,弃去孔内溶液,用洗涤缓冲液PBST洗三次,并用200μl封闭液在37℃封闭1小时。PBST洗5次后,将生物素标记的ssDNA适配子用SHCMK结合缓冲液稀释至5μg/ml。在94℃变性5min,并室温冷却15min后,取100μlssDNA适配子与靶菌体37℃孵育40min,用SHCMKT洗涤缓冲液洗涤5次。加入1:1000稀释的链酶亲和素标记的辣根过氧化物酶100μl,37℃孵育30min,并PBST洗涤6次。然后每孔加100μlTMB显色液,在37℃作用5min显色。用50μl终止液终止显色反应,酶标仪于450nm处测定OD值。
在28个测试的适配体中,ELISA测试OD值超过1.0的12个适配子被认为对胞内分枝杆菌具有高亲和力。鸟-胞内分枝杆菌复合群主要包括鸟分枝杆菌和胞内分枝杆菌,两者在进化上具有非常高的同源性。亲和性检测显示12个适配子对鸟分枝杆菌同样具有很高的亲和性,可以作为MAC的特征性适配子,其中MAC-A11适配子亲和性最高。
MAC-A11适配子的核苷酸序列为GGGAGCTCAGAATAAACGCTCAACCGGATCGCGCGATAGACTAGACGTGTTACGTTTGTTCGACATGAGGCCCGGATC(SEQ ID NO.1),二级结构如图1所示。
实施例2
本实施例应用MAC-A11适配子构建检测体系,并检测临床菌株,主要是选择具有高亲和性的MAC特异性适配子MAC-A11或适配子组合构建基于适配子的三明治夹心ELISA检测体系,用于95株临床菌株的检测(其中MAC共31株,MTB为53株和非分枝杆菌为11株);具体的步骤和结果如下:
(1)酶标板经30W 75cm的紫外灯照射处理12h。适配子用分光光度计测浓度,并经94℃变性5min,冰浴10min进行预处理;处理后的适配子用包被缓冲液(0.05mol/L碳酸盐缓冲液,pH值9.6)稀释,按照每孔1μg的浓度作为捕获适配子包被酶联板,4℃过夜;然后3%BSA(牛血清白蛋白)37℃下封闭1h;
(2)将菌体样品用PBS稀释至10μg/mL,以100μL/孔加入封闭后的酶标板,37℃孵育1h,加入PBST洗涤3次,3min/次;
(3)将5'端生物素标记的另一个适配子作为检测适配子,SELEX结合缓冲液稀释至0.5μg/孔加入酶标板,37℃,40min,SELEX洗涤缓冲液洗涤5次,3min/次;
(4)加入1:1000的链霉亲和素标记的辣根过氧化物酶100μl,37℃孵育30min;加入PBST洗涤5次,3min/次;
(5)显色试剂A与显色试剂B按照1:1的比例混合加入,37℃孵育10min,加入终止液终止显色;加入PBST洗涤3次,3min/次;
(6)利用酶标仪双波长(450nm和620nm)检测样本的吸光度值(A450,A620)。
结果判断:进行结果判断的OD值应为OD450nm与OD600nm的差值。阳性对照组(包括胞内分枝杆菌标准株ATCC 13950,鸟分枝杆菌标准株ATCC25291,和29株MAC临床菌株,其中鸟分枝杆菌临床菌株11株,胞内分枝杆菌临床菌株18株)OD值平均为0.26;阴性对照组平均OD值=0.06(包括53株MTB和11株非分枝杆菌,OD均值分别为0.08和0.05)。其中MAC组和结核分枝杆菌组之间的OD值差异有统计学意义(P<0.0001;AUC=0.9884,95%CI:0.9729-1.004);结核分枝杆菌组和非分枝杆菌组之间的OD值差异有统计学意义(P<0.0001;AUC=1.000,95%CI:1.000-1.000),参考图2。
综上所述,应用本发明中MAC适配子MAC-A11组合构建的检测体系可以特异性地检测出结核分枝杆菌菌株,非结核分枝杆菌和非分枝杆菌。本发明的MAC配子用于细菌学检测能特异性地检出MAC菌株。
实施例4
本实施例采用FITC标记MAC适配子MAC-A11,荧光显微镜下观察其检测临床菌株的效果,具体的实验过程和结果如下:
待测菌株:鸟分枝杆菌标准株ATCC 25291,胞内分枝杆菌标准株ATCC13950,3种结核分枝杆菌复合群菌株(标准株H37Rv,无毒株H37Ra,卡介苗BCG),2种非分枝杆菌(大肠杆菌,金黄色葡萄球菌)。
将适配子MAC-A11用FITC荧光标记,并经94℃变性5min,冰浴15min进行预处理;用500μL的PBS混匀处理后的适配子和不同菌体(2×107CFU),37℃温和震荡作用40min,12000rpm离心5min,用ddH2O洗涤沉淀四次,沉淀重悬于ddH2O中,分别取10μL沉淀涂片在荧光显微镜观察。
由图3可知,MAC标准株在荧光显微镜下均可见较强的绿色荧光,结核分枝杆菌和非分枝杆菌在显微镜下均未观测到绿色荧光。
以上对本发明的具体实施例进行了详细描述,但其只作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
序列表
<110> 上海市肺科医院
上海孚清生物科技有限公司
<120> 一种特异性识别鸟-胞内分枝杆菌复合群的DNA适配子及其筛选方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 78
<212> DNA
<213> Artificial Sequence (人工序列)
<400> 1
gggagctcag aataaacgct caaccggatc gcgcgataga ctagacgtgt tacgtttgtt 60
cgacatgagg cccggatc 78
Claims (4)
1. 一种特异性识别鸟-胞内分枝杆菌复合群的DNA适配子,其特征在于,所述DNA适配子的序列为SEQ ID NO. 1。
2.如权利要求1所述的DNA适配子在制备识别鸟-胞内分枝杆菌复合群试剂盒中的应用,其特征在于,所述试剂盒包括所述DNA适配子。
3.如权利要求1所述的DNA适配子在制备MAC肺病诊断试剂盒中的应用,其特征在于,所述试剂盒包括所述DNA适配子。
4.根据权利要求2或3所述的应用,其特征在于,所述DNA适配子采用生物素、荧光素或酶标记。
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WO2019234196A1 (en) * | 2018-06-06 | 2019-12-12 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | In vitro method for the diagnosis or detection of non-tuberculous mycobacteria |
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WO2019234196A1 (en) * | 2018-06-06 | 2019-12-12 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | In vitro method for the diagnosis or detection of non-tuberculous mycobacteria |
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