CN109234414B - 结核分枝杆菌的对氨基水杨酸耐药性诊断标志物及其应用 - Google Patents

结核分枝杆菌的对氨基水杨酸耐药性诊断标志物及其应用 Download PDF

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CN109234414B
CN109234414B CN201810695692.4A CN201810695692A CN109234414B CN 109234414 B CN109234414 B CN 109234414B CN 201810695692 A CN201810695692 A CN 201810695692A CN 109234414 B CN109234414 B CN 109234414B
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李海成
陈亮
郭卉欣
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Abstract

本发明公开了一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物，由Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c组成，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。本发明公开了结核分枝杆菌的对氨基水杨酸耐药性诊断标志物可实现结核分枝杆菌对氨基水杨酸是否耐药的快速临床检测，并为治疗对氨基水杨酸耐药提供了潜在靶点。

Description

结核分枝杆菌的对氨基水杨酸耐药性诊断标志物及其应用
技术领域
本发明涉及一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物及其应用。
背景技术
耐药结核病是全球结核病防控工作中面临的重大难题，尤其是耐多药结核病和广泛耐药结核病严重威胁人类身体健康，2013年报告显示虽然新发结核病患者减少，但耐多药结核病的诊治远远未达到预期目标。耐药结核病的及时有效监测是结核病防治工作的关键，目前我国结核病实验室对耐药结核病的诊断包括传统比例法药敏、BACTEC MGIT 960系统以及基于分子生物学的快速诊断方法等。
现有检测结核分枝杆菌对氨基水杨酸耐药的技术只有基于固体培养的比例法药敏检测，尚未有成熟的针对对氨基水杨酸耐药的快速分子生物学检测方案。现有技术的缺点主要包括：⑴耗时长，需要建立在分离培养的基础上进行药敏检测，分离培养需要4-8周时间，在此基础上进行后续的传统固体比例法(4-8周)或者液体药敏(1-2周)的时间；⑵场地限制，两种培养方法均需要体积较的培养箱，为保证生物安全需要固定的培养房间；⑶相对于利福平、异烟肼而言，尚未有针对对氨基水杨酸耐药的分子检测靶点及相关成熟的分子诊断产品。
发明内容
本发明的目的在于一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物及其应用。
本发明所采取的技术方案是：
一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物，由Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c组成。
进一步地，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。
一种用于检测结核分枝杆菌对氨基水杨酸耐药性的试剂盒，该试剂盒中含有定量Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量的试剂。
进一步地，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。
一种检测结核分枝杆菌对氨基水杨酸耐药性的方法，包括以下步骤：
1)定量待测结核分枝杆菌中Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量；
2)根据Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量，判断结核分枝杆菌对氨基水杨酸耐药性。
进一步地，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。
本发明的有益效果是：
本发明公开了结核分枝杆菌的对氨基水杨酸耐药性诊断标志物可实现结核分枝杆菌对氨基水杨酸是否耐药的快速临床检测，并为治疗对氨基水杨酸耐药提供了潜在靶点。
附图说明
图1为五个基因相关蛋白分子的互作网络图。
具体实施方式
基于目前的技术水平，本发明以对氨基水杨酸耐药新的分子标识为目标，以体外对氨基水杨酸梯度浓度药物筛选出的对照清晰的对氨基水杨酸耐药家系为研究对象，通过基因组甲基化水平、表达谱水平两个组学的检测，筛选出与对照组相比甲基化水平升高最明显同时表达水平明显降代的五个基因作为对氨基水杨酸耐药的诊断标志物。本发明的分子标志物来源于两个组学水平的筛选，通过组学筛选可以全面的找到有意义的分子标志物，但是会增加误选的机会，为解决这个难题，我们选用分子表达过程中紧密联系的两个生物学过程(甲基化、基因转录)进行数据的相互验证，找出新的、差异最明显且两组学数据理论上互相支持的基因作为对氨基水杨酸耐药的诊断标志物。
本发明中筛选诊断标志物的实验步骤如下：
(1)耐药家系的构建
首先挑选H37Rv单克隆菌株，扩增后保存为G0代菌株，同时准备好1麦氏浊度的菌液接种到对氨基水杨酸的梯度培养基上(培养基中对氨基水杨酸的含量分别为20、2-1、2-2、2-3、2-4μg/μL)，37℃培养箱中培养4周左右，取有菌落生长且菌量满足后续的冻存及传代的最高药物浓度培养基的菌株为G1代，以此类推，直至最高药浓度(WHO标准耐药浓度)培养上有足量细菌生长，定义为对氨基水杨酸耐药株模型构建成功。
(2)组学分析
扩增对氨基水杨酸耐药菌株与平行对照菌析，分别提取基因组、RNA进行后续的甲基化组学、转录组学测序分析，经生物信息分析后筛选出甲基化水平升高最显著且表达水平降低最显著的五个基因作为对氨基水杨酸耐药诊断的新的分子标志物。组合目前成熟的各种基因检测方法即可开发出新的对氨基水杨酸耐药快速诊断试剂盒。
经过两组学的联合分析，最终筛选到在对氨基水杨酸耐药过程甲基化水平升高同时表达水平降低的最显著的五个基因Rv3890c、Rv2002、Rv1886c、Rv3824c和Rv3825c，五个基因的表达量如下表所示。经过蛋白分子互作分析，得到五个基因相关蛋白分子的互作网络图(如图1所示)，显示五个基因均在结核分枝杆菌中有重要地位。
Figure BDA0001713482250000031
实施例1
一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物，由Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c组成，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。
实施例2
一种用于检测结核分枝杆菌对氨基水杨酸耐药性的试剂盒，该试剂盒中含有定量Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量的试剂，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。
实施例3
一种检测结核分枝杆菌对氨基水杨酸耐药性的方法，包括以下步骤：
定量待测结核分枝杆菌中Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量；
根据Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量，判断结核分枝杆菌对氨基水杨酸耐药性，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量时，判断结核分枝杆菌对对氨基水杨酸耐药。

Claims (4)

1.一种结核分枝杆菌的对氨基水杨酸耐药性诊断标志物，其特征在于，所述标志物由Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c组成。
2.根据权利要求1所述的结核分枝杆菌的对氨基水杨酸耐药性诊断标志物，其特征在于：当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量以及甲基化水平高于正常甲基化水平时，判断结核分枝杆菌对对氨基水杨酸耐药。
3.一种用于检测结核分枝杆菌对氨基水杨酸耐药性的试剂盒，其特征在于：该试剂盒中含有定量Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量和甲基化水平的试剂。
4.根据权利要求3所述的试剂盒，其特征在于，当Rv3890c、Rv2002、Rv1886c、Rv3824c、Rv3825c表达量低于正常表达量以及甲基化水平高于正常甲基化水平时，判断结核分枝杆菌对对氨基水杨酸耐药。
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