CN111218449A - 一种结核分枝杆菌核酸适配子及其制备方法 - Google Patents
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Abstract
本发明涉及适配子技术领域，公开了一种结核分枝杆菌核酸适配子，通过SELEX筛选方法得到结核分枝杆菌特异亲和的适配子，其核苷酸序列SEQ ID NO：0所示；对其进行结构改造，筛选出SEQ ID NO：2‑3。还公开了该种结核分枝杆菌核酸适配子的制备方法。本发明通过SELEX技术筛选出结核分枝杆菌高度特异亲和的核酸适配子，对适配子进行结构改造，提高对菌株结合的亲和性与特异性，利用荧光显微分析仪观察结核分枝杆菌的形态，并进行计数，可以显著提高菌种分析的灵敏度。此方法操作方法简单，对操作人员要求低，可以有效节约人力成本。
Description
技术领域
本发明涉及适配子技术领域，涉及了一种结核分枝杆菌核酸适配子，还包括该种结核分枝杆菌核酸适配子的制备方法。
背景技术
结核分枝杆菌(M.tuberculosis)是结核病的病原菌，可侵犯全身各组织器官，但以肺部感染最多见。1980年代后，随着艾滋病的流行、结核分枝杆菌耐药菌株的出现、免疫抑制剂的应用、吸毒、贫困及人口流动加剧等因素，全球范围内结核病的疫情骤然恶化。据WHO统计，全世界约每4个人中就有1个人感染了结核分枝杆菌，在某些发展中国家成人中结核分枝杆菌携带率高达80％，其中约5％～10％携带者可发展为活动性结核病。目前全球每年约出现1000万结核新病例，并导致约170万人死亡。我国每年死于结核病的人约25万之多，是各类传染病死亡人数总和的两倍多。因此，结核病又成为了威胁人类健康的全球性卫生问题，并成为某些发展中国家和地区，特别是艾滋病高发区人群的首要死因。世界卫生组织和各国对结核病的发展形势日益严峻重视。相比之下，人类对非结核分枝杆菌(NTM)所引起的感染，却未充分认识，也未被引起足够重视。从我国历次的结核病流行病学调查资料显示，NTM分离率由1990年的4.9％上升至2000年的11.1％，到2010年的22.9％，基本反映了我国的NTM病呈明显上升的态势，已成为威胁人类健康的重要公共卫生问题。更为重要的是，NTM肺病临床症状与体征极似肺结核病，同样可产生咳嗽、咯痰、咯血、胸痛、气急、盗汗、低热、乏力、消瘦、萎靡不振等症状，在无菌种鉴定结果的情况下，可长期被误诊为结核病。但两种感染的治疗用药却完全不同，NTM对许多抗结核药物天然耐药，且不同NTM菌种对药物敏感性各不相同。目前常用痰标本结核分枝杆菌检测方法是痰涂片镜检法，其检测灵敏度低(10000条/mL)，往往会导致部分病人会漏诊，无法在第一时间获得治疗而流向社会。由于结核分枝杆菌空气传播的特性，在人群中必然成为无法识别、无法防范的传染源。另外镜检法只能确认是否感染分枝杆菌，无法鉴别结核分枝杆菌还是非结核分枝杆菌，容易造成误诊与错误用药，错过最佳的治疗时间。痰结核分枝杆菌常规培养法即通过在鉴别培养基上培养后，通过观察细菌菌落形态、生长速度、生长温度和色素，以及一系列的生化试验，实现MTB和NTM的初步鉴定。该方法是鉴定细菌活性的“黄金标准”，但是操作比较繁琐、费时，需要4～8周时间，并受到菌量、菌龄的影响。此外，由于菌株易发生变异，出现非典型生物学特征，难以判断生化反应结果，而影响鉴定的准确性。以基因检测为诊断依据的结核分枝杆菌菌种鉴定方法包括Souther blotter、PCR扩增、RFLP、基因芯片、测序等。目前商品化的分枝杆菌菌种鉴定试剂盒都是以16S rDNA序列为检测靶标的。然而由于分枝杆菌某些菌种生长比较慢，16S rDNA保守性比较强，以单一的16SrDNA为检测靶标时，需进一步借助探针或测序的方法才能实现结核分枝杆菌和非结核分枝杆菌的鉴别诊断，而且操作复杂，检测成本高，降低了在临床上的广泛应用。
发明内容
本发明针对现有技术的缺点，提供了一种结核分枝杆菌核酸适配子，还公开了该种结核分枝杆菌核酸适配子的制备方法。
为了解决上述技术问题，本发明通过下述技术方案得以解决：
一种结核分枝杆菌核酸适配子，通过SELEX筛选方法得到结核分枝杆菌特异亲和的适配子，其核苷酸序列SEQ ID NO：0所示。SEQ ID NO：0为GGGAGCTCAGTTTAAACGCTCAATAGTCAAGCGATTGATAGACGGGCTTTGACACGTGTTCGAAGGGCATGGGGGATC。
作为优选，对其进行结构改造，筛选出SEQ ID NO：2-3。对SEQ ID NO：0进行结构改造，验证改造后适配子的灵敏度和特异性，并判断适配子在临床样本中的检测效果，筛选出SEQ ID NO：2-3。SEQ ID NO：2-3为CAATAGTCAAGCTTTGACACGTATGGGGG。
以上提到的结核分枝杆菌核酸适配子的制备方法，其特征在于：包括以下步骤，
通过构建单链寡核苷酸文库，利用SELEX技术筛选H37Rv适配子，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；对获得的单个适配子SEQ ID NO：0进行结构改造，获得4个序列与二级结构均不同的适配子，SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4；对4个适配子进行灵敏度检测，对灵敏度最高的适配子SEQ ID NO：2进行二次改造，获得4个序列与二级结构均不同的适配子SEQ ID NO：2-1、SEQ ID NO：2-2、SEQ IDNO：2-3、SEQ ID NO：2-4，检测其对结核分枝杆菌标准株H37Rv结合的灵敏度与特异性，获得2个灵敏度和特异性均良好的适配子SEQ ID NO：2-2、SEQ ID NO：2-3，用于临床痰液样本中结核分枝杆菌的检测，获得灵敏度最高的适配子SEQ ID NO：2-3。
通过构建单链寡核苷酸文库，具体步骤如下：设计合成78碱基对的单链DNA文库：5’-GGGAGCTCAGTTTAAACG-N35-CGAAGGGCATGGGGGATC-3’，其中N代表碱基ATGC中的任意一个，文库容量大约为1015～1016，并进一步得到纯化的单链ssDNA文库，用于H37Rv适配子的SELEX技术筛选；单链ssDNA文库中间为40个碱基的随机序列，两端为18个碱基的固定序列；构建上游引物：5’-GGGAGCTCAGTTTAAACG-3’，构建下游引物5’-CGAAGGGCATGGGGGATC-3。文库容量大约为1015～1016。
利用SELEX技术筛选H37Rv适配子，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；具体步骤如下：用于包被缓冲液将结核分枝杆菌标准株H37Rv包被于微孔板中，同时设空白反筛孔、非结核分枝杆菌和非分枝杆菌反筛孔；ssDNA文库和SELEX结合缓冲液混匀后先与空白反筛孔进行孵育，然后转移到H37Rv包被孔进行孵育，SELEX冲洗缓冲液洗涤，甩干后加入SELEX洗脱缓冲液与H37Rv结合的ssDNA，产物经酚氯仿抽提、乙醇沉淀纯化，PCR扩增后，进行10轮筛选，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；本发明通过SELEX技术筛选出结核分枝杆菌高度特异亲和的核酸适配子，对适配子进行结构改造，提高对菌株结合的亲和性与特异性，利用荧光显微分析仪观察结核分枝杆菌的形态，并进行计数，可以显著提高菌种分析的灵敏度。此方法操作方法简单，对操作人员要求低，可以有效节约人力成本。
通过SELEX技术筛选出结核分枝杆菌标准株H37Rv适配子，荧光标记后，可以应用适配子对结核分枝杆菌标准株H37Rv进行荧光显微检测。
通过对适配子的结构改造提高了适配子对结核分枝杆菌标准株H37Rv检测的灵敏度，并且可以应用于临床痰液样本中对结核分枝杆菌的检测。
具体实施方式
下面结合实施例对本发明作进一步详细描述。
实施例1
一种结核分枝杆菌核酸适配子，通过SELEX筛选方法得到结核分枝杆菌特异亲和的适配子，其核苷酸序列SEQ ID NO：0所示。
对其进行结构改造，筛选出SEQ ID NO：2-3。
以上提到的结核分枝杆菌核酸适配子的制备方法，包括以下步骤，
通过构建单链寡核苷酸文库，利用SELEX技术筛选H37Rv适配子，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；对获得的单个适配子SEQ ID NO：0进行结构改造，获得4个序列与二级结构均不同的适配子，SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4；对4个适配子进行灵敏度检测，对灵敏度最高的适配子SEQ ID NO：2进行二次改造，获得4个序列与二级结构均不同的适配子SEQ ID NO：2-1、SEQ ID NO：2-2、SEQ IDNO：2-3、SEQ ID NO：2-4，检测其对结核分枝杆菌标准株H37Rv结合的灵敏度与特异性，获得2个灵敏度和特异性均良好的适配子SEQ ID NO：2-2、SEQ ID NO：2-3，用于临床痰液样本中结核分枝杆菌的检测，获得灵敏度最高的适配子SEQ ID NO：2-3。
表一适配子结构改造
实施例2
一种结核分枝杆菌核酸适配子，通过SELEX筛选方法得到结核分枝杆菌特异亲和的适配子，其核苷酸序列SEQ ID NO：0所示。
对其进行结构改造，筛选出SEQ ID NO：2-3。
以上提到的结核分枝杆菌核酸适配子的制备方法，包括以下步骤，
步骤1
菌株制备：将结核分枝杆菌标准株H37Rv和非结核分枝杆菌转种至含有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，灭活30min。灭活后转至磨菌管，磨菌后调整浊度至1mg/mL。
步骤2
A.构建单链寡核苷酸文库：设计合成78碱基对的单链DNA文库：5’-GGGAGCTCAGTTTAAACG-N35-CGAAGGGCATGGGGGATC-3’，其中N代表碱基ATGC中的任意一个，文库容量大约为1015～1016，并进一步得到纯化的单链ssDNA文库，用于H37Rv适配子的SELEX技术筛选；
B.利用SELEX技术筛选H37Rv适配子：用于包被缓冲液将结核分枝杆菌标准株H37Rv包被于微孔板中，同时设空白反筛孔、非结核分枝杆菌和非分枝杆菌反筛孔；ssDNA文库和SELEX结合缓冲液混匀后先与空白反筛孔进行孵育，然后转移到H37Rv包被孔进行孵育，SELEX冲洗缓冲液洗涤3遍，洗去未结合的ssDNA，甩干后加入SELEX洗脱缓冲液与H37Rv结合的ssDNA，产物经酚氯仿抽提、乙醇沉淀纯化，PCR扩增后，进行10轮筛选，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；
C.(1)对获得的单个适配子SEQ ID NO：0进行结构改造，获得4个序列与二级结构均不同的适配子，SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4；对4个适配子用FAM荧光标记，用0.05mol/L碳酸盐溶解至1μM，取1μg与H37Rv菌体(2×107CFU)于500μLPBS中37℃温和振荡作用40min。将菌体-适配子复合物12000rpm离心沉淀10min，并用500μLPBS洗涤两次。最终将沉淀重悬于20μLddH2O中，并涂匀于载玻片上。在荧光显微镜下观察发出的绿色荧光，将光学显微镜下观察结果作为对照，与SEQ ID NO：0作对比，结果显示SEQ IDNO：2对结核分枝杆菌标准株H37Rv识别的灵敏度最高。
(2)第二次适配子结构改造与灵敏度、特异性验证：对灵敏度最高的适配子SEQ IDNO：2进行二次改造，获得4个序列与二级结构均不同的适配子SEQ ID NO：2-1、SEQ ID NO：2-2、SEQ ID NO：2-3、SEQ ID NO：2-4。对上述4个适配子用FAM荧光标记，用0.05mol/L碳酸盐溶解至1μM，取1μg分别与H37Rv标准株、NTM菌体(2×107CFU)于500μL PBS中37℃温和振荡作用40min。将菌体-适配子复合物12000rpm离心沉淀10min，并用500μLPBS洗涤两次。最终将沉淀重悬于20μLddH2O中，并涂匀于载玻片上。在荧光显微镜下观察发出的绿色荧光，将光学显微镜下观察结果作为对照。结果显示SEQ ID NO：2-2、SEQ ID NO：2-3的灵敏度与特异性均良好。
(3)SEQ ID NO：2-2、SEQ ID NO：2-3应用于临床痰液样本中结核分枝杆菌的检测。
痰标本处理：根据标本的粘稠程度，加1～2倍体积(一般约5ml)的4％NaOH，涡旋振荡器振荡，使标本均匀液化。37℃水浴15min后加入1/15M pH6.8磷酸缓冲液至40～45mL并混匀，离心3000g，15min，去其上清液；沉淀物中再次加入磷酸缓冲液至40～45mL并混匀，离心3000g，15min，去其上清液；将用800μL磷酸缓冲液重悬沉淀，混匀，-20℃保存。
取上述处理后的痰标本200μL至新的1.5mL离心管中，12000rpm离心5min，弃上清。用200μL1×PBS重悬沉淀，离心洗涤，12000rpm，5min，弃上清。再次用200μL 1×PBS重悬沉淀。
将FAM标记的适配子94℃变性5min，并室温冷却15min。取1μM适配子加入于上述200μL沉淀重悬液中，37℃温和振荡作用40min，12000rpm离心沉淀10min，并用200μL PBS洗涤3-5次。最终将沉淀重悬于20μLddH2O中，并涂匀于载玻片上。在荧光显微镜下观察发出的绿色荧光，将光学显微镜下观察结果作为对照。结果显示SEQ ID NO：2-3在临床痰液样本中的检测的灵敏度更好。
总之，以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所作的均等变化与修饰，皆应属本发明专利的涵盖范围。
序列表
上海孚清生物科技有限公司
一种结核分枝杆菌核酸适配子及其制备方法
9
1
78
DNA
SEQ ID NO0
1
GGGAGCTCAGTTTAAACGCTCAATAGTCAAGCGATTGAT 39
AGACGGGCTTTGACACGTGTTCGAAGGGCATGGGGGATC 39
2
58
DNA
SEQ ID NO1
2
CAATAGTCAAGCGATTGATAGACGGGCTT 29
TGACACGTGTTCGAAGGGCATGGGGGATC 29
3
43
DNA
SEQ ID NO2
3
CAATAGTCAAGCTTTGACACGTGTTCGAAGGGCATGGGGGATC 43
4
50
DNA
SEQ ID NO3
4
AAGCGATTGATAGACGGGCTTTGAC 25
ACGTGTTCGAAGGGCATGGGGGATC 25
5
40
DNA
SEQ ID NO4
5
CAATAGTCAAGCGATTGATAGACGGGCTTTGACACGTGTT 40
6
43
DNA
SEQ ID NO2-1
6
CAATAGTCAAGCTTTGACACGTGTTCGAAGAACATGGGGGATC 43
7
32
DNA
SEQ ID NO2-2
7
CAATAGCACGTGTTCGAAGAACATGGGGGATC 32
8
32
DNA
SEQ ID NO2-3
8
CAATAGTCAAGCTTTGACACGTATGGGGGATC 32
9
21
DNA
SEQ ID NO2-4
9
CAATAGCACGTATGGGGGATC 21
Claims (3)
1.一种结核分枝杆菌核酸适配子，其特征在于：通过SELEX筛选方法得到结核分枝杆菌特异亲和的适配子，其核苷酸序列SEQ ID NO：0所示。
2.根据权利要求1所述的结核分枝杆菌核酸适配子，其特征在于：对其进行结构改造，筛选出SEQ ID NO：2-3。
3.权利要求1所述的结核分枝杆菌核酸适配子的制备方法，其特征在于：包括以下步骤，
通过构建单链寡核苷酸文库，利用SELEX技术筛选H37Rv适配子，筛选后的饱和文库，经克隆测序，获得单个适配子SEQ ID NO：0；对获得的单个适配子SEQ ID NO：0进行结构改造，获得4个序列与二级结构均不同的适配子，SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQID NO：4；对4个适配子进行灵敏度检测，对灵敏度最高的适配子SEQ ID NO：2进行二次改造，获得4个序列与二级结构均不同的适配子SEQ ID NO：2-1、SEQ ID NO：2-2、SEQ ID NO：2-3、SEQ ID NO：2-4，检测其对结核分枝杆菌标准株H37Rv结合的灵敏度与特异性，获得2个灵敏度和特异性均良好的适配子SEQ ID NO：2-2、SEQ ID NO：2-3，用于临床痰液样本中结核分枝杆菌的检测，获得灵敏度最高的适配子SEQ ID NO：2-3。
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