CN105063188B - 结核杆菌感染检测的荧光原位杂交检测试剂盒 - Google Patents
结核杆菌感染检测的荧光原位杂交检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种用于肺结核病理组织切片中结核杆菌感染检测的荧光原位杂交检测试剂盒。用于结核杆菌感染检测的荧光原位杂交检测试剂盒，其组成包括：杂交液、变性液，DAPI复染剂，和分隔并集中包装这些试剂瓶或管的包装盒。所述的结核杆菌荧光原位杂交检测试剂盒杂交液含有三种特异探针，分别为IS6110探针，其序列如SEQ ID NO：1所示；MPRT40探针，其序列如SEQ ID NO：2所示；和16s rRNA探针，其序列如SEQ ID NO：3所示。
Description
技术领域
本发明属于医学检测领域，具体涉及一种用于结核杆菌感染检测的荧光原位杂交检测试剂盒。
背景技术
结核病被列为我国重大传染病之一，是严重危害人民群众健康的呼吸道传染病。卫生部全国第五次结核病流行病学抽样调查结果显示，目前我国结核病年发病人数约为130 万，占全球发病的14.3%，位居全球第2位。2001～2010 年，全国共发现和治疗肺结核患者828万例，其中传染性肺结核患者450万例。
传统上诊断肺结核，有三种途径：（1）痰液及病变组织中查出结核杆菌；（2）结核的病理组织常规检查；（3）胸部X线检查，但该方法不能确诊肺结核，因为肺部其他疾病，如肺炎、肺寄生虫病、肺部肿瘤等病变也可出现类似肺结核病变的肺部X线影像学表现，单凭X线检查，许多病人会误诊误治，不但给病人造成经济上的损失，还会导致病情恶化。痰液及病变组织结核杆菌阳性一般来说是肺结核最可靠的诊断，称为金标准。但是由于检出率低，如果单独以痰液结核杆菌检查作为唯一的诊断标准会造成大部分结核患者的漏诊。
病理学诊断在疾病诊断中的价值逐渐体现出来，变得越来越重要。目前结核病的病理诊断主要靠组织学的特殊形态改变和抗酸染色。组织学存在病变形态不典型、与结节病、肺癌、肺炎等病理组织学形态相似的情况，而且某些细菌的感染也会引起类似结核病的组织形态，因此容易误诊。抗酸染色由于针对的是所有抗酸杆菌，特异性不强，染色阳性者还须与其它非致病的抗酸杆菌进行鉴别，而且受实验条件的影响很大，如脱蜡不净、酒精、初染温度、背景等，很容易出现结果的假阴性，造成误诊。长期以来由于诊断技术的不足，全国约有 50%的肺结核病人未能及时确诊及时治疗，给健康人群构成巨大威胁。因此，建立一种可以辅助病理医生从疑似肺结核患者的肺活检组织中快速、明确诊断出是否存在结核杆菌感染的方法就显得尤为必要。
发明内容
本发明的目的在于克服现有技术的不足，提供一种高灵敏度、高可信度同时又非常直观的结核杆菌感染检测试剂盒及其检测方法。
本发明通过以下技术方案实现上述目的：
一种用于结核杆菌荧光原位杂交检测的分子探针；分别为IS6110探针，其序列如SEQ ID NO：1所示；MPRT40探针，其序列如SEQ ID NO：2所示；和16s rRNA其序列如SEQ IDNO：3所示。
用于结核杆菌感染检测的荧光原位杂交检测试剂盒，其组成包括：杂交液、DAPI复染剂，和分隔并集中包装这些试剂瓶或管的包装盒。
在本发明的一个实施方案中，所述杂交液含有荧光标记的三种特异探针，分别为IS6110探针，其序列如SEQ ID NO：1所示；MPRT40探针，其序列如SEQ ID NO：2所示；和16srRNA探针，其序列如SEQ ID NO：3所示；三种探针的浓度分别为10-50pM；进一步优选为30pM。
在本发明优选的实施方案中，所述杂交液的具体组成为：10%(w/v)硫酸葡聚糖，10mM NaCl，20% (v/v)甲酰胺，0.1% (w/v)焦磷酸钠，0.2% (w/v)聚乙烯基吡咯烷酮，5mMNa2EDTA，0.1% (v/v)TritonX-100，50mM Tris/HCl(pH7.5)；分别为30pM的所述荧光标记的三种特异探针。
所述DAPI复染剂的主要成分为DAPI（4,6-二脒基-2-苯吲哚盐酸）和抗褪色液。
每次检测都必须同时进行质控片操作，质控片必须和病例样本一同操作。质控片可以购买商品化对照片或者选择已知的对照样本自行制备。
本发明的试剂盒能够用于检测的样本范围广泛，包括但不限于，痰、咽拭子、胃灌洗液、支气管灌洗液、活体组织、吸引物、咳出物、体液(脊髓、胸腹水、心包液等)、血液、脓液、骨髓、尿液、组织切片、食物样本、来自土壤、空气和水的样本，以及它们的培养物。这些样本经相应处理后，原则上是只要保持细胞形态完整并且靶核酸未被破坏，均可使用本发明的试剂盒进行检测。这些样本的处理方法是本领域技术人员所掌握的，例如：
痰：痰液涂片法；
脓液：同痰液涂片法；
病灶组织：对病灶组织进行切片；
尿液：留全量夜尿，静置4～5h后，弃上清液，取沉淀部分尿液10ml，3000rpm，离
心30min，取沉淀物涂片；
胸、腹水标本：参照尿液涂片法；
脑脊液：无菌操作收集脑脊液，放置冰箱或室温24h，待薄膜形成后涂片。也可将脑脊液离心沉淀，3000rpm，离心30min，弃上清液，取沉淀物涂片。
本发明与现有技术相比具有如下优点：
（1）能直观的将检测结核杆菌与其引起的病理变化结合起来；
（2）在高效、准确检测结核杆菌的同时可由样本病理切片上分析感染率和感染强度，这是其他分子生物学检测方法所不具有的特点；
（3）由于杂交过程中探针与IS6110、MPRT40和16s rRNA的杂交是特异的，且信号是用荧光标记的，所以较常规的染色观察检测灵敏、准确；
（4）本发明杂交显色后的病理切片可长期保存。
附图说明
图1.结核杆菌感染的肺结核病理组织切片杂交检测结果。
具体实施方式
下面将结合附图以及进一步的详细说明来举例说明本发明。需要指出的是，以下说明仅仅是对本发明要求保护的技术方案的举例说明，并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
实施例1特异性探针的制备
根据NCBI报道的人源结核杆菌基因序列，进行同源性比对分析，查找该基因的最保守区域，选取IS6110、MPRT40和16s rRNA基因作为靶序列，结合运用primer5.0、oligo6.0和Primer express2.0软件，设计出三组结合杆菌特异性荧光探针。将探针放入NCBI作BLAST进行同源性分析，结果显示与其他基因序列异源，从而避免了杂交时非特异性的存在。探针由生工生物工程（上海）有限公司合成。
所述的三条寡核苷酸探针的序列分别如SEQ ID NO：1-3所示。
荧光标记探针的制备：选用FITC荧光素，采用缺口平移法进行探针的直接标记。将荧光素直接与探针核苷酸以磷酸戊糖骨架共价结合。具体步骤如下：
① 使用TE缓冲液将PCR纯化产物溶解成终浓度为1g/L。
② 制备含1μl浓度是3 mg/ml的DNA酶储存液及10×DNA酶溶液，体积为1ml。
③ 冰浴中配制缺口平移反应体系，加样完成后混匀，37℃静置12 min。具体反应体系如下：
| 探针DNA | 8μl |
| 10×NT缓冲液 | 10μl |
| 10μg/mlBSA | 5μl |
| 10μg/ml DNA酶Ⅰ溶液 | 10μl |
| dNTP(含有dATP、dGTP、dCTP各1mM) | 5μl |
| FITC（终浓度为60-70μM） | 8μl |
| 灭菌三蒸水 | 54μl |
| 反应总体积 | 100μl |
④ 加入2.5μl浓度为10U/μl的大肠杆菌DNA聚合酶。
⑤ 置PCR扩增仪，15℃孵育12个小时，70℃变性5min 4℃5小时。
⑥ 加入4μl 的0.2M EDTA( pH=8.0)终止反应，乙醇沉淀，最后用cot-1DNA悬浮沉淀粉末。
⑦ TAE琼脂糖凝胶电泳检测探针标记结果，对标记后的产物作纯化处理并测定其OD值，通过公式计算出荧光素的掺入率及探针的浓度。
⑧保存：加等体积甘油，-20℃分装存放。
实施例2检测试剂盒的制备
杂交液组成：10％(w/v)硫酸葡聚糖，10mM NaCl，20％(v/v)甲酰胺，0.1％(w/v)焦磷酸钠，0.2％(w/v)聚乙烯基吡咯烷酮，5mM Na2EDTA，0.1％(v/v)TritonX-100，50mMTris/HCl(pH7.5)；分别为30pM的荧光标记的三种特异探针（实施例1制备）。
DAPI复染剂的制备：取2.2mgDAPI粉末，加入550μl蒸馏水混匀，制成DAPI原液4mg/ml。4℃保存。工作液：取2.5μlDAPI原液加入5ml蒸馏水中，混匀，2μg/ml。
实施例3检测试剂盒的应用
分别取同一病人的痰液样本和病灶组织样本，共20病例。
痰液样本采用罗氏培养法，具体为选用改良的Lowenstein-Jensen 培养基，将处理过的痰液进行培养。具体为取痰标本1mL 加入4％氢氧化钠2mL，涡旋震荡30 秒混匀，室温静置20min，其间振荡2～3次，促痰液化。以灭菌刻度毛细吸管取0.1mL 消化后痰液，缓缓均匀地接种每支培养基斜面上，放37℃培养箱内培养。接种后第3 天和第7 天观察培养的情况，之后每周观察1 次，直到第8周末。若出现培养阳性则随时报告，培养至8 周未见细菌生长，报告为培养阴性。
病灶组织样本采用本发明实施例2制备的试剂盒进行检测，具体方法如下：
1、玻片预处理
玻片放入65±5℃恒温箱中烤片过夜；取出，放入二甲苯中室温脱蜡15分钟；放入另一缸二甲苯中室温继续脱蜡15分钟；放入无水乙醇中室温10分钟，去除残留二甲苯；再放入100%、90%、70%梯度乙醇室温复水各3分钟；放入灭菌纯化水中室温洗涤3分钟，用无绒纸巾吸取多余水分；再将其放入灭菌纯化水中100±5℃煮片20分钟（切片水平放置于容器中，样本面朝上）；取出玻片，室温晾干；放入37±1℃预热的蛋白酶K反应液中消化20分钟；放入2×SSC中室温洗涤5分钟；再放入另一缸2×SSC中室温洗涤5分钟；取出，依次放入70%，90%，100%梯度乙醇室温脱水各3分钟；取出玻片，室温晾干。
2、样品和探针同时变性（避光操作）
加10μl的杂交液到杂交区域，迅速盖上盖玻片，轻压使杂交液均匀分布，避免产生气泡；用橡皮胶沿盖玻片边缘封片，完全覆盖盖玻片和载玻片接触的部位；将玻片放在78℃的热台上，变性5分钟；将玻片放入预热的湿盒或杂交盒中，避光，37±1℃孵育过夜（10-18小时）。
3、 杂交后洗涤及复染（避光操作）
迅速将玻片放入预热的杂交盒中，避光，37±1℃孵育过夜（约16小时）。玻片放入洗液中37±1℃孵育 10-15分钟；取出玻片，再将其放入室温70%乙醇中脱水3分钟；取出玻片，暗处自然干燥玻片；室温，滴加10 μl DAPI复染剂到的盖玻片, 载玻片目标区域朝下，轻放于盖玻片上，轻压，避免产生气泡，在暗处存放，待观察。
4、镜检：结核分枝杆菌阴性(-) ：连续观察50 个不同视野，未发现结核分枝杆菌。
结核分枝杆菌阳性(报告杆菌数) ：1-9 条/50 视野。
结核分枝杆菌阳性(1+) ：10-99 条/50 视野。
结核分枝杆菌阳性(2+) ：1-9 条每视野。
结核分枝杆菌阳性(3+) ：10-99 条每视野。
结核分枝杆菌阳性(4+) ：>100 条每视野。
结果表明，痰液培养与本发明试剂盒检测结果完全一致。
实施例4试剂盒的特异性检测
取5种结核杆菌菌株和10种非结核杆菌菌株，采用实施例3的方法，用实施例2制备的试剂盒进行检测，具体结果如下：
本发明内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或多个技术方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
SEQUENCE LISTING
<110> 广州华银医学检验中心有限公司
<120> 结核杆菌感染检测的荧光原位杂交检测试剂盒
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
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<213> 人工合成
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cggtcggaag ctcctatgac aatgcactag ccgagacgat caacggccta tacaagaccg 180
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Claims (2)
1.一种用于结核杆菌荧光原位杂交检测的检测试剂盒，其特征在于，其组成包括：杂交液、变性液，DAPI复染剂，和分隔并集中包装这些试剂瓶或管的包装盒；
所述杂交液含有三种特异探针，分别为IS6110探针，其序列如SEQ ID NO：1所示；MPRT40探针，其序列如SEQ ID NO：2所示；和16s rRNA其序列如SEQ ID NO：3所示；三种探针的浓度分别为0.1-0.5μM；
所述杂交液的具体组成为：10%(w/v)硫酸葡聚糖，10mM NaCl，20% (v/v)甲酰胺，0.1%(w/v)焦磷酸钠，0.2% (w/v)聚乙烯基吡咯烷酮，5mM Na2EDTA，0.1% (v/v)TritonX-100，50mM Tris/HCl(pH7.5)；分别为30pM的所述荧光标记的三种特异探针。
2.根据权利要求1所述的检测试剂盒，其特征在于，所述DAPI复染剂中DAPI（4,6-二脒基-2-苯吲哚盐酸）的浓度为4mg/ml。
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