WO2017173698A1 - 用于诊断结核分枝杆菌感染的分子标志物、引物组及应用 - Google Patents

用于诊断结核分枝杆菌感染的分子标志物、引物组及应用 Download PDF

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WO2017173698A1
WO2017173698A1 PCT/CN2016/081402 CN2016081402W WO2017173698A1 WO 2017173698 A1 WO2017173698 A1 WO 2017173698A1 CN 2016081402 W CN2016081402 W CN 2016081402W WO 2017173698 A1 WO2017173698 A1 WO 2017173698A1
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曾谷城
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中山大学
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  • the invention belongs to the field of biotechnology, and particularly relates to a molecular marker for diagnosing Mycobacterium tuberculosis infection and its application in the diagnosis of Mycobacterium tuberculosis infection.
  • Tuberculosis is a kind of "high infection rate”, “high pathogenicity rate” and “high mortality rate” caused by Mycobacterium tuberculosis (Mtb) infection.
  • WHO World Tuberculosis Annual Report released by the World Health Organization (WHO) in 2015, the number of deaths from tuberculosis reached 1.5 million a year, which has exceeded the number of deaths caused by AIDS or HIV infection.
  • China is one of the countries with the largest number of M. tuberculosis carriers, the largest number of active tuberculosis patients, and the most serious tuberculosis-resistant epidemic. Therefore, it is extremely urgent to quickly control the spread of tuberculosis.
  • tuberculosis infection can provide a basis for clinical treatment and medication, in order to provide timely and rapid public health prevention and control measures for the tuberculosis control department to provide decision-making basis.
  • the current diagnostic techniques for Mycobacterium tuberculosis infection mainly include bacteriological diagnosis and immunological diagnosis.
  • the bacteriological diagnosis includes Mycobacterium tuberculosis sputum smear acid-fast staining, Mycobacterium tuberculosis culture, and Mycobacterium tuberculosis specific nucleic acid PCR detection of sputum specimens.
  • the bacteriological diagnosis technique of Mycobacterium tuberculosis based on acid-fast staining, culture, and PCR of sputum specimens relies heavily on the presence of Mycobacterium tuberculosis in the sputum discharged from tuberculosis patients.
  • tuberculosis cannot detect M. tuberculosis in sputum because they do not have sputum or sputum, and M. tuberculosis cannot be detected in sputum, and M. tuberculosis not only has a very long culture period, but often It takes 3 to 6 weeks and the culture requirements are high. Therefore, the bacteriological diagnosis technology of Mycobacterium tuberculosis infection has great limitations, and has the defects of low diagnostic efficiency, long cycle, low sensitivity, complicated technology, and can not be applied to clinically >50% of sputum specimens and tuberculosis branches. Diagnosis of bacilli-negative tuberculosis patients.
  • Immunological diagnostic techniques for tuberculosis mainly include ELISA or ELISPOT detection of tuberculin skin test (TST) and IFN- ⁇ release.
  • TST tuberculin skin test
  • the main advantage of the tuberculin skin test (TST) is the relatively inexpensive, TB screening for large-scale tuberculosis infections.
  • diagnosis of the tuberculin skin test (TST) lacks M. tuberculosis specificity, and the results of the tuberculin skin test (TST) are very susceptible to non-specific interference with the body's immunity and physiological state. Therefore, the tuberculin skin test (TST) has a fatal defect of poor diagnostic specificity and low sensitivity.
  • Tuberculosis immunological diagnostic techniques based on ELISA or ELISPOT detection of M.
  • tuberculosis-specific IFN- ⁇ release have higher specificity than tuberculin skin test (TST) diagnostic techniques.
  • TST tuberculin skin test
  • tuberculosis-free IFN- ⁇ release ELISA or ELISPOT detection of tuberculosis The epidemiological diagnosis technology requires a professional cell culture laboratory to culture the cells of the subject. The cell culture experiment period is long. The ELISPOT data reading requires a professional and expensive ELISPOT spot reading instrument. The overall diagnosis is expensive and does not apply to the grassroots. The early diagnosis of tuberculosis is not suitable for the diagnosis of tuberculosis in infants and young children.
  • tuberculosis patients are in an immunosuppressive or disorder state, and the expression of IFN- ⁇ is suppressed or in a disordered state. Therefore, there is a technically natural defect in the tuberculosis diagnosis technique based on tuberculosis-specific IFN- ⁇ release.
  • the first aspect of the present invention provides a novel molecular marker for diagnosing M. tuberculosis infection, the molecular marker being selected from the sequences of the RNAs shown in SEQ ID NOS. One or a combination of two or more.
  • the molecular marker is selected from RNA having the same sequence or >50% identical sequence as any of the fragments of one of the RNAs set forth in SEQ ID NOS: 1 to 28; or the molecular marker is SEQ ID NO Any one of the RNA full lengths or fragments shown in Figures 1-28 having artificially synthesized, spliced, recombinant or cloned RNA with >50% RNA sequence similarity.
  • a second aspect of the invention provides the use of a molecular marker as described above for the preparation of a medicament for the diagnosis of a Mycobacterium tuberculosis infection.
  • the preparation is used to diagnose the subject by detecting the content of the molecular marker in the peripheral blood, peripheral blood, peripheral blood serum or peripheral blood mononuclear cells (PBMC) of the subject, and comparing with the normal level. Whether the person is infected with M. tuberculosis. More specifically, it can be detected by real-time quantitative PCR or a gene chip whether or not the subject is infected with M. tuberculosis.
  • PBMC peripheral blood mononuclear cells
  • a third aspect of the present invention provides a primer set comprising at least one primer pair or two or more primers for specifically amplifying a primer pair of RNAs represented by SEQ ID NOS. 1 to 28, respectively.
  • a combination of pairs wherein the primer pairs for the specific amplification sequences such as the RNAs shown in SEQ ID NOS. 1 to 5, 7-17, 19-24, and 26-28 are sequentially primer pairs 1 to 5, 7 -17, 19-24, 26-28, a primer pair for specifically amplifying an RNA such as SEQ ID NO. 6 is a primer pair 6a or 6b for a specific amplification sequence such as SEQ ID NO.
  • the primer pair of RNA is the primer pair 18a or 18b
  • the primer pair for specifically amplifying the RNA of the sequence such as SEQ ID NO. 25 is the primer pair 25a or 25b.
  • Primer pairs can be natural or synthetic.
  • a fourth aspect of the invention provides the use of the above primer set for the preparation of a preparation for diagnosing a Mycobacterium tuberculosis infection.
  • a fifth aspect of the present invention provides a preparation for diagnosing a Mycobacterium tuberculosis infection, which comprises at least one of a primer pair for specifically amplifying an RNA as shown in SEQ ID NOS. 1 to 28, respectively.
  • Primer pair or a combination of two or more primer pairs.
  • the primer pairs of the RNA are respectively the primer pairs 1 to 5, 7-17, 19-24, and 26-28 described above, and the primer pair for specifically amplifying the RNA of the sequence such as SEQ ID NO.
  • a primer pair for specifically amplifying a sequence such as the RNA of SEQ ID NO. 18 is a primer pair 18a or 18b
  • a primer pair for specifically amplifying a sequence such as the RNA of SEQ ID NO. 25 is Primer pair 25a or 25b.
  • the preparation may be a kit or a gene chip or the like.
  • a sixth aspect of the present invention provides a preparation for diagnosing Mycobacterium tuberculosis infection, comprising: 1) total RNA extraction reagent of peripheral blood mononuclear cells (PBMC); 2) reverse transcription reagent; 3) real-time quantitative PCR The reagent; wherein the real-time quantitative PCR reagent comprises at least one primer pair for a specific amplification sequence such as the RNA shown in SEQ ID NOS. 1 to 28, or a combination of two or more primer pairs. Further preferably, the primer pair for specifically amplifying the RNA as shown in SEQ ID NOS. 1 to 5, 7-17, 19-24, and 26-28 is the primer pair 1 described above.
  • PBMC peripheral blood mononuclear cells
  • primer pairs for specifically amplifying the RNA as shown in SEQ ID NO. 6 are primer pairs 6a or 6b, for specific amplification sequences such as SEQ
  • the primer pair of the RNA shown by ID NO. 18 is the primer pair 18a or 18b
  • the primer pair for specifically amplifying the RNA as shown in SEQ ID NO. 25 is the primer pair 25a or 25b.
  • the total RNA extraction reagent and the reverse transcription reagent of the peripheral blood mononuclear cells in the preparation can adopt the corresponding reagents existing in the art, for example, the retro transcription reagent is a reverse transcription reagent kit of Thermo, and the total RNA extraction reagent is extracted by a Trizol method. Common reagents required for RNA.
  • the preparation may be a kit or the like.
  • RNA of the present invention as shown in SEQ ID NOS. 1 to 28 is significantly lower or higher in tuberculosis patients infected with M. tuberculosis than in M. tuberculosis.
  • the RNAs represented by the sequences as shown in SEQ ID NOS. 1 to 28 can be used as specific molecular markers or diagnostic targets for discriminating whether or not they are infected by M. tuberculosis, and can be used for preparation for diagnosis of tuberculosis.
  • the use of the molecular marker of the present invention to develop a preparation for diagnosing Mycobacterium tuberculosis infection is useful for diagnosing Mycobacterium tuberculosis infection, and is sensitive, convenient, rapid, specific, and inexpensive.
  • FIG. 1 Human peripheral blood mononuclear cells (PBMC) in a healthy population of M. tuberculosis-infected tuberculosis patients or in a healthy population not infected with M. tuberculosis, as shown by SEQ ID NOS. 1 to 8, 10 to 17 a real-time quantitative PCR result map of differential expression;
  • PBMC peripheral blood mononuclear cells
  • Figure 2 Human peripheral blood mononuclear cells (PBMC) of a healthy population of M. tuberculosis-infected tuberculosis patients or a healthy population not infected with M. tuberculosis by RNA as shown in SEQ ID NO. 18-25, 27-28 A graph of real-time quantitative PCR results of differential expression in .
  • PBMC peripheral blood mononuclear cells
  • Figure 3 Differential expression of RNA as shown in SEQ ID NO. 9, 26 in human peripheral blood mononuclear cells (PBMC) of M. tuberculosis-infected tuberculosis patients or healthy populations not infected with M. tuberculosis Real-time quantitative PCR results plot.
  • PBMC peripheral blood mononuclear cells
  • tuberculosis patients infected with M. tuberculosis 27 cases, abbreviated as TB group
  • healthy people not infected with M. tuberculosis 26 cases, abbreviated as Healthy group
  • Real-time quantitative PCR was used to detect the expression of 28 RNAs in each of the peripheral blood mononuclear cells (PBMC) (the sequence of which is shown in SEQ ID NO. 1-28), and the above 28 RNAs were found in tuberculosis patients infected with M. tuberculosis.
  • the level of expression is generally significantly lower or higher than that of healthy people who are not infected with M. tuberculosis. Specific steps are as follows:
  • Step 1 Preparation of suspension of peripheral blood mononuclear cells (PBMC):
  • PBS phosphate buffer
  • the specific operation the above cell suspension was aspirated into a centrifuge tube, centrifuged at 8000 g for 4 minutes at 4 ° C, and the supernatant was discarded, taking care not to destroy the cell pellet; 1 ml of Trizol was added to each tube and repeated with a sample gun. Blow until no obvious precipitation, let stand for 5 minutes at room temperature; add chloroform (one-fifth volume of Trizol) to the homogenate lysate, mix until the solution is milky white, let stand for 5 minutes at room temperature; centrifuge at 12000g for 15 minutes at 4 °C.
  • the reverse transcription reaction system was prepared on ice using Thermo's reverse transcription kit, as shown in Table 1:
  • the reaction system was set up and placed in a common PCR reaction apparatus.
  • the procedure was 25 ° C for 5 min ⁇ 42 ° C for 60 min ⁇ 70 ° C for 5 min. After the reaction was completed, it was stored at 4 ° C.
  • Step 4 Fluorescence quantitative PCR reaction
  • the KPA Biosystems fluorescent quantitative PCR kit was used, and the reaction system is shown in Table 2 below:
  • the reaction system was prepared according to Table 2, and a real-time quantitative PCR reaction was carried out using a Bio-Rad IQ5 instrument.
  • Standard procedure for amplification of real-time quantitative PCR reactions 95 ° C for 3 minutes; followed by 95 ° C for 3 seconds, 60 ° C for 30 seconds, 40 cycles.
  • the GAPDH gene is used as an internal reference gene.
  • Primers designed to specifically amplify 28 RNAs sequences as shown in SEQ ID NO. 1-28), as well as specific amplification primers for the GAPDH gene.
  • the primer pair sequences for specifically amplifying the RNA or gene shown in SEQ ID NO. 1-28, respectively are shown in Table 3 below (primers are synthesized by the Huada gene), wherein the specific amplification sequence is used.
  • the primer pair of the RNA as shown in SEQ ID NO. 6 may be the primer pair 6a or 6b
  • the primer pair for specifically amplifying the RNA as shown in SEQ ID NO. 18 may be the primer pair 18a or 18b.
  • a primer pair for a specific amplification sequence such as the RNA shown in SEQ ID NO. 25 may be primer pair 25a or 25b.
  • the pair of primers with the primer pair number 1 is called the primer pair 1
  • the pair of primers with the number 2 is called the primer pair 2, and so on.
  • the primer sequences specific for the internal reference gene GAPDH gene are as follows:
  • RNAs corresponding to the sequences shown in SEQ ID NOS. 1 to 8, 10 to 25, and 27 to 28 in PBMC of tuberculosis patients infected with M. tuberculosis are generally significantly lower than those in the PBMCs of tuberculosis patients infected with M. tuberculosis. Healthy people who are not infected with M. tuberculosis.
  • the expression level of RNA corresponding to the sequence shown in SEQ ID NO. 9, 26 in the PBMC of M. tuberculosis-infected tuberculosis patients was significantly higher than that in the healthy population not infected with M. tuberculosis.
  • the sequence of the present invention such as the RNAs shown in SEQ ID NOS. 1 to 28, was significantly lower in the tuberculosis patients infected with M. tuberculosis than the healthy persons not infected with M. tuberculosis. Therefore, the RNAs of the sequences as shown in SEQ ID NOS. 1 to 28 can be used as specific molecular markers or diagnostic targets for discriminating whether or not they are infected by M. tuberculosis, and these molecular markers and specific primers thereof can be applied.
  • a preparation for diagnosing M. tuberculosis infection For the preparation of a preparation for diagnosing M. tuberculosis infection.

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Abstract

本发明提供用于诊断结核分枝杆菌感染的分子标志物，选自序列如SEQ ID NO.1-28所示的RNA中的一种或两种以上的组合；或者选自与SEQ ID NO.1-28所示的其中任何一种RNA的片段具有相同序列或者>50%相同序列的RNA；或者是与SEQ ID NO.1-28所示的其中任何一种RNA全长或片段具有>50%的RNA序列相似性的人工合成、拼接、重组或克隆的RNA。本发明还提供所述分子标志物的特异性引物组，以及所述分子标志物及引物组在制备用于诊断结核分枝杆菌感染的制剂中的应用。

Description

用于诊断结核分枝杆菌感染的分子标志物、引物组及应用 技术领域
本发明属于生物技术领域，特别涉及用于诊断结核分枝杆菌感染的分子标志物及在结核分枝杆菌感染诊断中的应用。
背景技术
结核病(Tuberculosis,TB)是因结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染引起的一种“高感染率”、“高致病率”与“高致死率”烈性传染病。2015年世界卫生组织(WHO)发布的世界结核年报显示，结核病的年致死人数达到150万，已经超过因艾滋病或HIV感染所致死人数。中国是全球结核分枝杆菌携带人数最多、活动性结核病人最多以及结核耐药疫情最为严重的国家之一。所以，迅速控制结核疫情蔓延迫在眉睫。
早期、快速、便捷诊断结核是控制结核病的一个关键环节。只有早期、快速、便捷、准确诊断出是否被结核分枝杆菌感染，才能为临床治疗用药提供依据，才能为结核病控制部门采取及时、快速的公共卫生防控措施提供决策依据。
目前的结核分枝杆菌感染诊断技术主要包括有细菌学诊断与免疫学诊断。其中细菌学诊断包括结核分枝杆菌痰涂片抗酸染色、结核分枝杆菌培养、及痰标本的结核分枝杆菌特异核酸PCR检测等。但是，基于痰标本抗酸染色、培养、PCR的结核分枝杆菌细菌学诊断技术严重依赖于结核病人排出的痰液里面必须有结核分枝杆菌。然而，有＞50％的结核感染病人因不排痰或者排的痰里面不含结核分枝杆菌所以无法在痰液里面检测到结核分枝杆菌，而且结核分枝杆菌不仅培养周期非常漫长，往往需要3～6周，而且培养要求高。所以，结核分枝杆菌感染的细菌学诊断技术具有很大的局限性，存在诊断效率低、周期长、灵敏度低、技术复杂等缺陷，而且无法适用于临床约＞50％的痰标本结核分枝杆菌阴性结核病人的诊断。
结核的免疫学诊断技术主要包括结核菌素皮试(TST)与IFN-γ释放的ELISA或者ELISPOT检测。结核菌素皮试(TST)的主要优点是价格相对低廉、适宜大规模结核感染人群的结核筛查。但是，结核菌素皮试(TST)的诊断缺乏结核分枝杆菌特异性，而且结核菌素皮试(TST)的结果非常容易受到机体免疫力与生理状态的非特异性干扰。所以结核菌素皮试(TST)存在诊断特异性差、灵敏度低的致命缺点。基于结核分枝杆菌特异的IFN-γ释放的ELISA或者ELISPOT检测的结核免疫学诊断技术相比结核菌素皮试(TST)诊断技术有更高的特异性。但是，基于结核特异的IFN-γ释放ELISA或者ELISPOT检测的结核免 疫学诊断技术需要专业的细胞培养实验室对受试者的细胞进行培养，细胞培养实验周期长，ELISPOT的数据读出需要专业、昂贵的ELISPOT斑点读出仪器，总体诊断价格昂贵，不适用于基层的结核早期、快速诊断，也不适合婴幼儿的结核诊断。而且，大量的结核病人处于免疫抑制或紊乱状态，IFN-γ的表达受到抑制或者处于紊乱的状态，所以，基于结核特异的IFN-γ释放的结核诊断技术存在技术上天然的缺陷。
因此，发现新的结核特异的诊断生物标记物，开发灵敏、方便、快捷、特异性高并且廉价的结核诊断新技术就成为了控制结核疫情蔓延的重要工作。
发明内容
为弥补现有技术的不足，本发明第一方面提供用于诊断结核分枝杆菌感染的新型分子标志物，所述分子标志物选自序列如SEQ ID NO.1～28所示的RNA中的一种或两种以上的组合。
或者，所述分子标志物选自与SEQ ID NO.1～28所示的任何其中一种RNA的片段具有相同序列或者＞50％相同序列的RNA；或者所述分子标志物是与SEQ ID NO.1～28所示的任何其中一种RNA全长或片段具有＞50％的RNA序列相似性的人工合成、拼接、重组或克隆的RNA。
本发明第二方面提供上文所述的分子标志物在制备用于诊断结核分枝杆菌感染的制剂中的应用。作为一种具体方式，该制剂通过检测被试者末梢血、外周血、外周血血清或外周血单个核细胞(PBMC)中所述分子标志物的含量，并与正常水平相比较来诊断被试者是否被结核分枝杆菌感染。进一步具体的，可通过实时定量PCR或基因芯片来检测判别被试者是否被结核分枝杆菌感染。
本发明第三方面提供一种引物组，该引物组包括分别用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种引物对或两种以上引物对的组合，其中，用于特异性扩增序列如SEQ ID NO.1～5、7-17、19-24、26-28所示的RNA的引物对分别依次为引物对1～5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6的RNA的引物对为引物对6a或6b，用于特异性扩增序列如SEQ ID NO.18的RNA的引物对为引物对18a或18b，用于特异性扩增序列如SEQ ID NO.25的RNA的引物对为引物对25a或25b。引物对可以是天然的或是合成的。
本发明第四方面提供上述引物组在制备用于诊断结核分枝杆菌感染的制剂中的应用。
本发明第五方面提供一种用于诊断结核分枝杆菌感染的制剂，该制剂包括分别用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种引物对、或两种以上引物对的组合。进一步，用于特异性扩增序列如SEQ ID NO.1～5、7-17、19-24、26-28所示 的RNA的引物对分别依次为上文所述的引物对1～5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6的RNA的引物对为引物对6a或6b，用于特异性扩增序列如SEQ ID NO.18的RNA的引物对为引物对18a或18b，用于特异性扩增序列如SEQ ID NO.25的RNA的引物对为引物对25a或25b。该制剂可以是试剂盒或基因芯片等。
本发明第六方面提供一种用于诊断结核分枝杆菌感染的制剂，该制剂包括：1)外周血单个核细胞(PBMC)的总RNA提取试剂；2)逆转录试剂；3)实时定量PCR试剂；其中，所述实时定量PCR试剂包括用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种引物对、或两种以上引物对的组合。进一步优选的，用于特异性扩增序列如SEQ ID NO.1～5、7-17、19-24、26-28所示的RNA的引物对分别依次为上文所述的引物对1～5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6所示的RNA的引物对为引物对6a或6b，用于特异性扩增序列如SEQ ID NO.18所示的RNA的引物对为引物对18a或18b，用于特异性扩增序列如SEQ ID NO.25所示的RNA的引物对为引物对25a或25b。该制剂中的外周血单个核细胞的总RNA提取试剂和逆转录试剂均可采用本领域现有的相应试剂，如逆转录试剂采用Thermo公司的逆转录试剂盒，总RNA提取试剂采用Trizol法提取RNA所需的常用试剂。该制剂可以是试剂盒等。
经实验证实，本发明所述的序列如SEQ ID NO.1～28所示的RNA在被结核分枝杆菌感染的结核病患者中的表达量明显低于或高于未被结核分枝杆菌感染的健康人，因而，所述的序列如SEQ ID NO.1～28所示的RNA可以作为判别是否被结核分枝杆菌感染的特异分子标志物或诊断靶点，可应用于制备用于诊断结核分枝杆菌感染的制剂中。采用本发明的分子标志物开发诊断结核分枝杆菌感染的制剂，用于诊断结核分枝杆菌感染，具有灵敏、方便、快捷、特异性高、廉价的特点。
附图说明
图1：为序列如SEQ ID NO.1～8、10～17所示的RNA在结核分枝杆菌感染的结核病患者或未被结核分枝杆菌感染的健康人群的人外周血单个核细胞(PBMC)中差异表达的实时定量PCR结果图；
图2：为序列如SEQ ID NO.18～25、27～28所示的RNA在结核分枝杆菌感染的结核病患者或未被结核分枝杆菌感染的健康人群的人外周血单个核细胞(PBMC)中差异表达的实时定量PCR结果图。
图3：为序列如SEQ ID NO.9、26所示的RNA在结核分枝杆菌感染的结核病患者或未被结核分枝杆菌感染的健康人群的人外周血单个核细胞(PBMC)中差异表达的实时定量PCR结果图。
具体实施方式
下面结合附图和实施例对本发明的技术方案做进一步说明：
实施例
本实施例将人群分为两个组：结核分枝杆菌感染的结核病患者(27例，简写为TB组)、未被结核分枝杆菌感染的健康人群(26例，简写为Healthy组)；通过实时定量PCR检测每例外周血单个核细胞(PBMC)中28种RNA(其序列如SEQ ID NO.1-28所示)的表达情况，发现结核分枝杆菌感染的结核病患者中上述28种RNA的表达水平总体明显低于或高于未被结核分枝杆菌感染的健康人群。具体步骤如下：
步骤一：外周血单个核细胞(PBMC)悬液的制备：
取肝素抗凝静脉血3ml与等量的磷酸缓冲液(PBS)充分混匀得混合液；另取离心管，加入等量的淋巴细胞分离液；用吸管将上述混合液沿着离心管管壁缓慢叠加于淋巴细胞分离液液面上，保持液面清楚；水平离心，1500rpm×20分钟，用吸管小心吸取中间白膜层于新的离心管中，加入5倍体积的PBS，1500rpm×10分钟洗涤细胞两次，末次离心去除上清，加入含10％(v/v)胎牛血清的RPMI1640，重悬细胞，每例取10微升进行计数。
步骤二：RNA提取
采用Trizol法提取RNA，具体操作：将上述细胞悬液吸入离心管中，8000g 4℃离心2分钟，弃上清，注意不要破坏细胞沉淀；向每管中加入1ml的Trizol，用加样枪反复吹打至无明显沉淀，室温静置5分钟；向匀浆裂解液中加入氯仿(Trizol的五分之一体积)，混匀至溶液成乳白色，室温静置5分钟；12000g 4℃离心15分钟，分三层，吸取上清到新的离心管中；向上清中加入二分之一Trizol体积的异丙醇，上下颠倒混匀，室温静置10分钟，12000g 4℃离心10分钟；弃上清，加入Trizol等量的75％(v/v)的乙醇，轻轻上下颠倒清洗管壁，7500g 4℃离心5分钟后，弃上清；打开离心管盖，室温干燥几分钟，加入20～100微升的RNase-free水溶解沉淀。于-80℃保存待用。
步骤三：逆转录
采用Thermo公司的逆转录试剂盒，逆转录反应体系配制在冰上进行，具体如下表1：
Figure PCTCN2016081402-appb-000001
Figure PCTCN2016081402-appb-000002
按上述表1中配好反应体系，放入普通PCR反应仪器内，程序为25℃5min→42℃60min→70℃5min，反应结束后放4℃保存。
步骤四：荧光定量PCR反应
采用Kapa Biosystems公司的荧光定量PCR试剂盒，反应体系如下表2：
Figure PCTCN2016081402-appb-000003
按照表2来配制反应体系，使用Bio-Rad IQ5仪器进行实时定量PCR反应。
实时定量PCR反应的扩增标准程序：95℃3分钟；之后95℃3秒，60℃30秒，40个循环。
以GAPDH基因为内参基因。设计用于特异性扩增28种RNA(序列如SEQ ID NO.1-28所示)的引物，以及GAPDH基因的特异性扩增引物。其中，分别用于特异性扩增序列如SEQ ID NO.1-28所示的RNA或基因的引物对序列如下表3所示(引物由华大基因合成)，其中用于特异性扩增序列如SEQ ID NO.6所示的RNA的引物对可为引物对6a或6b，用于特异性扩增序列如SEQ ID NO.18所示的RNA的引物对可为引物对18a或18b，用于特异性扩增序列如SEQ ID NO.25所示的RNA的引物对可为引物对25a或25b。表3中，引物对序号为1的引物对称为引物对1，序号为2的引物对称为引物对2，以此类推。
表3
Figure PCTCN2016081402-appb-000004
Figure PCTCN2016081402-appb-000005
Figure PCTCN2016081402-appb-000006
Figure PCTCN2016081402-appb-000007
特异性针对内参基因GAPDH基因的引物序列如下：
Figure PCTCN2016081402-appb-000008
Figure PCTCN2016081402-appb-000009
根据实时定量PCR的结果，使用GraphPad Prism5对TB组与Healthy组进行student t-test统计差异分析，P值＜0.05表示具有统计学差异，以GAPDH基因为内参基因，采用delta CT方法来计算出结核病人相对健康人群的各个RNA的表达量，结果如图1～3(图1-3中各图左上角的数字和序列表中所对应RNA的序列编号对应)所示，横坐标表示实验组名称，纵坐标表示基因相对表达量。
从图1～2中可知，结核分枝杆菌感染的结核病患者的PBMC中的对应于序列如SEQ ID NO.1～8、10～25、27～28所示的RNA的表达水平总体显著低于未被结核分枝杆菌感染的健康人群。从图3可知，结核分枝杆菌感染的结核病患者的PBMC中的对应于序列如SEQ ID NO.9、26所示的RNA的表达水平显著高于未被结核分枝杆菌感染的健康人群。
上述实验证实，本发明的序列如SEQ ID NO.1～28所示的RNA在被结核分枝杆菌感染的结核病患者中的表达量明显低于或高于未被结核分枝杆菌感染的健康人，因而，所述的序列如SEQ ID NO.1～28所示的RNA可以作为判别是否被结核分枝杆菌感染的特异分子标志物或诊断靶点，这些分子标志物及其特异性引物可应用于制备用于诊断结核分枝杆菌感染的制剂中。
文中所用的试剂若未特别说明均为市场购买获得，文中未特别说明之处均为本领域技术人员根据其掌握的公知常识或常规技术手段所能理解或知晓，不再一一赘述。
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (10)

  1. 用于诊断结核分枝杆菌感染的分子标志物，其特征在于，所述分子标志物选自序列如SEQ ID NO.1～28所示的RNA中的一种或两种以上的组合；
    或者，所述分子标志物选自与SEQ ID NO.1～28所示的任何其中一种RNA的片段具有相同序列或者＞50％相同序列的RNA；或者所述分子标志物是与SEQ ID NO.1～28所示的任何其中一种RNA全长或片段具有＞50％的RNA序列相似性的人工合成、拼接、重组或克隆的RNA。
  2. 权利要求1所述的分子标志物在制备用于诊断结核分枝杆菌感染的制剂中的应用。
  3. 引物组，其特征在于，所述引物组包括分别用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种，其中，用于特异性扩增序列如SEQ ID NO.1-5、7-17、19-24、26-28所示的RNA的引物对分别依次为引物对1-5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6、18、25所示的RNA的引物对分别依次为引物对6a或6b、引物对18a或18b、引物对25a或25b，引物对1～5、6a、6b、7-17、18a、18b、19-24、25a、25b、26-28的碱基序列如下表1所示：
    表1
    Figure PCTCN2016081402-appb-100001
    Figure PCTCN2016081402-appb-100002
  4. 权利要求3所述的引物组在制备用于诊断结核分枝杆菌感染的制剂中的应用。
  5. 一种用于诊断结核分枝杆菌感染的制剂，其特征在于，包括分别用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种。
  6. 根据权利要求5所述的制剂，其特征在于，用于特异性扩增序列如SEQ ID NO.1～5、7-17、19-24、26-28所示的RNA的引物对分别依次为权利要求3中所述的引物对1～5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6、18、25所示的RNA的引物对分别依次为权利要求3中所述的引物对6a或6b、引物对18a或18b、引物对25a或25b。
  7. 根据权利要求5所述的制剂，其特征在于，所述制剂为试剂盒或基因芯片。
  8. 一种用于诊断结核分枝杆菌感染的制剂，其特征在于，包括：1)外周血单个核细胞(PBMC)的总RNA提取试剂、2)逆转录试剂、3)实时定量PCR试剂；其中，所述实时定量PCR试剂包括分别用于特异性扩增序列如SEQ ID NO.1～28所示的RNA的引物对中的至少一种。
  9. 根据权利要求8所述的制剂，其特征在于，用于特异性扩增序列如SEQ ID NO.1～5、7-17、19-24、26-28所示的RNA的引物对分别依次为权利要求3中所述的引物对1～5、7-17、19-24、26-28，用于特异性扩增序列如SEQ ID NO.6、18、25所示的RNA的引物对分别依次为权利要求3中所述的引物对6a或6b、引物对18a或18b、引物对25a或25b。
  10. 根据权利要求8所述的制剂，其特征在于，所述制剂为试剂盒。
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