CN114859041A - 结核杆菌的检测方法与试剂 - Google Patents

结核杆菌的检测方法与试剂 Download PDF

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CN114859041A
CN114859041A CN202110147460.7A CN202110147460A CN114859041A CN 114859041 A CN114859041 A CN 114859041A CN 202110147460 A CN202110147460 A CN 202110147460A CN 114859041 A CN114859041 A CN 114859041A
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interferon
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mycobacterium tuberculosis
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逄宇
郭继东
高孟秋
张福真
田建
张洪静
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Beijing Chest Hospital
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Abstract

结核杆菌的检测方法与试剂。本发明公开了炎症因子在结核杆菌感染的患者中的高表达，尤其是在IGRAs试验结果呈假阴性的结核病患者中的高表达。这一重要发现使得IGRAS试验结果中假阴性的降低成为可能。

Description

结核杆菌的检测方法与试剂
技术领域
本发明涉及微生物学，具体涉及一种检测微生物的方法如结核杆菌的检测方法及相应的试剂
背景技术
结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb) 感染引起可侵入人体全身各种器官，但主要以侵犯肺脏为主的慢性传染性疾病，一直是全球公共卫生关注的问题。2019年，全球约有1000万例病例和141万例死亡病例归因于结核病，使其成为传染性疾病的主要死亡原因。中国的结核病负担在全球排名第三，仅次于印度和印度尼西亚。利用有效的控制策略，中国结核病的发病率在过去几十年持续下降。尽管在结核病控制和预防方面取得了巨大成就，但报告的病例中只有三分之一是细菌确诊的结核病[Global tuberculosis report 2020.Geneva:World Health Organization；2020.Licence:CC BY-NC-SA 3.0IGO；Du J,Shu W,Liu Y,Wang Y, Zhan Y,Yu K,Gao J,LiL,Pang Y:Development and validation of external quality assessment panels formycobacterial culture testing to diagnose tuberculosis in China.Eur J ClinMicrobiol Infect Dis 2019,38(10):1961-1968.]，这对于结核病的发现及治疗造成了障碍，因此临床迫切需要新的诊断方法来提高结核杆菌确诊的病例。
干扰素-γ释放试验(IGRASs)是通过检测Mtb特异性T细胞分泌IFN-γ来诊断结核潜伏感染的主要方法[Pai M,Zwerling A,Menzies D:Systematic review: T-cell-basedassays for the diagnosis of latent tuberculosis infection:an update.AnnIntern Med 2008,149(3):177-184.]，因为在BCG(卡介苗)接种人群中， IGRASs具有优于结核菌素皮肤试验的特异性。
干扰素-γ释放试验(IGRASs)尽管在活动性结核病诊断方面表现出有限的效用，但许多国家指南继续推荐使用IGRASs作为活动性结核的发现手段[Stephan C,Wolf T,Goetsch U,Bellinger O,Nisius G,Oremek G,Rakus Z,Gottschalk R,Stark S,Brodt HRet al:Comparing QuantiFERON-tuberculosis gold,T-SPOT tuberculosis andtuberculin skin test in HIV-infected individuals from a low prevalencetuberculosis country.AIDS 2008,22(18):2471-2479；Nicol MP,Davies MA,Wood K,Hatherill M,Workman L,Hawkridge A,Eley B,Wilkinson KA,Wilkinson RJ, HanekomWA et al:Comparison of T-SPOT.TB assay and tuberculin skin test for theevaluation of young children at high risk for tuberculosis in a communitysetting.Pediatrics 2009,123(1):38-43.]。特别是IGRASs阴性是排除活动性结核的指标；然而，以往的观察性研究显示，约8％-19％的培养阳性结核病患者的IGRAS结果为阴性[Sester M,Sotgiu G,Lange C,Giehl C,Girardi E,Migliori GB,Bossink A,Dheda K,Diel R,Dominguez J et al:Interferon-gamma release assays for the diagnosis ofactive tuberculosis:a systematic review and meta-analysis.Eur Respir J 2011,37(1):100-111.]。因此，越来越多的人担心，对于活动性结核病的诊断，特别是在诊断疾病和初始治疗时，对这些假阴性结果的不恰当解释将会造成误诊。
因此，进一步降低假阴性的发生率，是提高干扰素-γ释放试验(IGRASs)诊断准确率的必要手段，这将对临床活动性结核病的诊断具有重要意义。
发明内容
我们的研究发现结核杆菌感染的患者会出现一些炎症因子的高表达，尤其是在IGRAs试验结果呈假阴性的结核病患者中。这一重要发现使得IGRAs试验结果中假阴性的降低成为可能。
因此，第一方面，本发明提供一种诊断标记物，所述诊断标记物包括一系列炎症因子，在检测样品或检测部位中检测标记物可用于诊断患者是否感染结核分支杆菌，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的诊断标记物包含炎症因子IL10；进一步优选本发明的诊断标记物包含炎症因子IL10和IFN-γ。
进一步，本发明提供一种辅助IGRAs诊断患者结核分支杆菌感染的辅助诊断标记物，该辅助诊断标记物含有炎症因子，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素 (Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor, TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的辅助诊断标记物包含炎症因子IL10；进一步优选本发明的辅助诊断标记物包含炎症因子IL10和IFN-γ。
更进一步，本发明提供一种鉴别IGRAs检测阴性的患者是否有结核分支杆菌感染的鉴别诊断标记物，该鉴别诊断标记物含有炎症因子，所述炎症因子选自细胞因子、C 反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子 (Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的鉴别诊断标记物包含炎症因子IL10；进一步优选本发明的鉴别诊断标记物包含炎症因子IL10和IFN-γ。
第二方面，本发明提供一种用于诊断患者是否感染结核分支杆菌的诊断试剂盒，用于快速、便捷、及时地诊断患者是否有感染结核分支杆菌的风险，所述诊断试剂盒包含用于检测炎症因子的检测试剂和/或检测装置。
所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明用于诊断患者是否感染结核分支杆菌的诊断试剂盒包含检测炎症因子IL10的检测试剂和/或检测装置；进一步优选，本发明的用于诊断患者是否感染结核分支杆菌的诊断试剂盒包含检测炎症因子IL10和IFN-γ的检测试剂和/或检测装置。
进一步，本发明提供一种鉴别IGRAs检测阴性的患者是否有结核分支杆菌感染的鉴别诊断试剂盒，该鉴别诊断试剂盒包含用于检测炎症因子的检测试剂和/或检测装置。
所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明鉴别IGRAs检测阴性的患者是否有结核分支杆菌感染的鉴别诊断试剂盒包含检测炎症因子IL10的检测试剂和/或检测装置；进一步优选，本发明的用于鉴别IGRAs检测阴性的患者是否有结核分支杆菌感染的鉴别诊断试剂盒包含检测炎症因子IL10和IFN-γ的检测试剂和/或检测装置。
第三方面，本发明提供一种检测患者是否感染结核分支杆菌的方法，所述方法包括对炎症因子的检测，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的检测是否感染结核分支杆菌的方法包含对炎症因子IL10的检测；进一步优选，本发明的检测是否感染结核分支杆菌的方法包含对炎症因子IL10和 IFN-γ的检测。
进一步，本发明提供一种辅助IGRAs检测患者是否感染结核分支杆菌的检测方法，所述方法包括对炎症因子的检测，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素 (Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor, TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的辅助IGRAs检测患者是否感染结核分支杆菌的检测方法包含对炎症因子IL10的检测；进一步优选，本发明的辅助IGRAs检测患者是否感染结核分支杆菌的检测方法包含对炎症因子IL10和IFN-γ的检测。
更进一步，本发明提供一种鉴别IGRAs检测阴性的患者是否感染结核分支杆菌的检测方法，所述方法包括对炎症因子的检测，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的鉴别IGRAs检测阴性的患者是否感染结核分支杆菌的检测方法包含对炎症因子IL10的检测；进一步优选，本发明的鉴别IGRAs检测阴性的患者是否有结核杆菌感染的检测方法包含对炎症因子IL10和IFN-γ的检测。
第四方面，本发明提供一种炎症因子在制备检测患者是否感染结核分支杆菌的诊断试剂盒中的用途，所述诊断试剂盒包含用于检测炎症因子的检测试剂和/或检测装置。
所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的用于制备检测患者是否感染结核分支杆菌的诊断试剂盒用途的炎症因子包含IL10；进一步优选包含炎症因子IL10和IFN-γ。
进一步，本发明提供一种炎症因子在制备辅助IGRAs检测患者是否感染结核分支杆菌的辅助诊试剂盒中的用途，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin, IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的用于制备检测患者是否感染结核分支杆菌的辅助诊断试剂盒用途的炎症因子包含IL10；进一步优选包含炎症因子IL10和IFN-γ。
更进一步，本发明提供一种炎症因子在制备鉴别IGRs检测阴性的患者是否感染结核分支杆菌的鉴别诊断试剂盒中的用途，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合。进一步，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)等中的一种或两种以上因子的组合。
所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A等；
所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ等；
所述肿瘤坏死因子选自：TNF、TNFα、TNFβ等。
优选，本发明的用于制备检测患者是否感染结核分支杆菌的鉴别诊断试剂盒用途的炎症因子包含IL10；进一步优选包含炎症因子IL10和IFN-γ。
通过本发明检测试剂及检测方法的使用，能够显著减少IGRAs试验假阴性的发生，从而提高IGRAs试验诊断结核的效能。
附图说明
图1为：IL-10在IGRAs阴性结核患者外周血单个核细胞中的表达上调的图 A-C:Mtb抗原刺激PBMC后IL-10、TGF-β、IL-4 mRNA表达水平； D:Mtb抗原刺激PBMC上清中IL-10的水平。
具体实施方式
本发明中所述患者为哺乳动物，包括人类、家畜、宠物、实验动物等，其中人类包括各年龄段、各种性别特征的患者，进一步为首次检测或经多次结核杆菌检测的患者。
本发明中所述的炎症因子的检测样品为血液、体腔中的液体，所述体腔选自腹腔、盆腔、关节腔、胸腔环境和/或脑部腔隙等；所述体腔中的液体选自腹腔、盆腔、关节腔、胸腔环境中的液体和/或脑脊液。
本发明中结核杆菌侵入的部位包括肺部、胸膜、脑膜、腹膜、肠、皮肤、骨骼、淋巴等部位。
用于本发明的炎症因子的检测方法是现有技术已有的检测方法，包括炎症因子的分泌水平的检测或炎症因子核酸表达水平的检测，进一步包括炎症因子mRNA表达水平的检测，具体示例包括实时荧光定量检测方法、炎性因子蛋白水平ELISA分析、液相芯片方法、流式细胞分析方法等
在本发明中，检测IGRAs(-)患者，所述炎症因子的含量尤其是IL-10的含量为大于1.5pg/mL,优选大于1.7pg/mL，更进一步优选大于2.0pg/mL。
为了进一步说明本发明结核检测试剂产品和方法的效果，特给出如下实施例，所述实施例仅是本发明方法的一种示例，并不会对本发明保护主题及保护范围造成约束，在本发明思路范围内的其他等同技术也属于本发明的范畴。
实施例1：PBMCs分离和结核抗原肽共刺激
PBMCs分离和外周血巨噬细胞诱导，具体步骤如下：
(1)人群纳入标准：
IGRAs(+)结核病患者：患者结核分枝杆菌病原学证据阳性(涂片、培养、xpert) 且IGRAS阳性，无其他严重病史；
IGRAs(-)结核病患者：患者结核分枝杆菌病原学证据阳性(涂片、培养、xpert) 且IGRAs阴性，无其他严重病史；
排除标准：HIV合并感染以及合并其他疾病个体，以及表现出不确定IGRAs反应的任何人。
(2)取2组人群外周血3-4ml，置于含EDTA抗凝剂的紫帽采血管中，利用TBD人全血单个核细胞分离液(LDS1075，天津市灏洋生物制品科技有限责任公司)进行单个核细胞分离。
(3)将单个核细胞铺入24孔细胞培养板，每孔加入2×106个细胞，每孔加入500μl含有10％FBS、1％青霉素-链霉素(Invitrogen)，和4mM 1-谷氨酰胺的RPMI1640，在37℃，5％CO2条件下培养，予以ESAT-6和CFP-10各100ul共刺激24h后，5000RPM/Min, 收集细胞上清和细胞沉淀与EP管中冻存备用。
表1：一般人群特征
Figure BDA0002931146380000081
BMI：身体质量指数。计量资料均符合正态分布用t检验，均数±标准误表示；分类变量资料采用卡方检验。显著性水平:p＜0.05.
实施例2：实时荧光定量(QRT)PCR检测IL-10、TGF-β、IL-4mRNA表达水平
(A)上述步骤细胞沉淀
(B)细胞总RNA提取
(1)把细胞收集在无RNA酶的1.5ml EP管中，5000rpm，离心5min，吸弃上清。
(2)利用细胞/细菌总RNA提取试剂盒(R1061，广州东盛生物科技有限公司)提取细胞的总RNA。
(C)cDNA合成(翊圣生物试剂盒，HifairTMⅡ1st Stand cDNA Synthesis SuperMixfor Qpcr，产品编号11123ES50)
(1)逆转录反应体系配制(20μl)：在RNase free的八排管中加入2 x HifairTMⅡSuperMix plus，再加入步骤(B)提取的总RNA 10μl，用移液枪轻轻吹打混匀。
(2)逆转录程序设置：25℃5min；42℃30min；85℃5min。
(3)利用无菌水将逆转录的cDNA稀释5倍。
(D)qPCR反应(翊圣生物试剂盒，
Figure BDA0002931146380000082
qPCR SYBR Green Master Mix(Low RoxPlus))
反应体系(冰上配制)：
表2 qPCR反应体系
Figure BDA0002931146380000091
表3 qPCR扩增程序(两步法)
Figure BDA0002931146380000092
(E)所用引物序列
以上实施例2(D)所用引物序列：
表4 qPCR引物序列
Figure BDA0002931146380000093
(F)结果分析
我们分析了结核分枝杆菌刺激的PBMC抗炎细胞因子的转录水平。如图1中A-C所示，与IGRAs阳性组相比，IGRAs阴性组IL-10表达明显上调,IL-4、TGF-βmRNA水平 在两组间无明显差异。
实施例3:IGRAs阳性组和IGRAs阴性组外周血PBMC经结核抗原肽刺激24h后上清IL-10水平。
(A)ELISA检测细胞因子IL-10：
1.将试剂和样本平衡至室温。
2.准备各种ELISA需要的试剂，将待测样本上清稀释2倍。
3.稀释好知道浓度的标准品，将其于待测样本分别加入包被于抗原的96 孔板中，孵育2小时。
4.弃去上清后清洗4次，加入生物素化的二抗，孵育1小时。
5.弃去上清，清洗4遍。
6.加入辣根过氧化物酶的链霉亲和素，孵育45分钟。
7.弃去上清，清洗4遍。
8.加入显色剂，暗室继续孵育30分钟。
9.加入终止液，使用分光光度仪在450nm处测OD值。
10.绘制标准曲线，算待测样本浓度。
(B)实验结果分析
IGRAs阴性组细胞上清中IL-10的浓度为4.77pg/mL，高于IGRAS阳性组的1.47 pg/mL,P＝0.007。
在培养确认的结核病患者中，外周血单个核细胞中IL-10的表达增加与干扰素-γ释放试验阴性结果相关。因此我们提出IL-10和IFN-γ联合检测可以显著提高IGRAs 检测的特异性，极大的降低其假阴性的发生率。
虽然本发明以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (8)

1.一种用于诊断患者是否感染结核分支杆菌的诊断标记物，其特征在于：所述诊断标记物包括一系列炎症因子，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合，优选所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)中的一种或两种以上因子的组合。
2.根据权利要求1所述的用于诊断患者是否感染结核分支杆菌的诊断标记物，其特征在于：所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A；所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ；所述肿瘤坏死因子选自：TNF、TNFα、TNFβ。
3.一种用于诊断患者是否感染结核分支杆菌的试剂盒，其特征在于：包含用于检测权利要求1-2中任一项所述标记物的检测试剂和/或检测装置。
4.一种权利要求1-2任一项所述标记物在用于制备诊断患者是否感染结核分支杆菌的检测试剂或检测试剂盒中的用途，所述试剂盒包含用于检测所述标记物的检测试剂或检测装置。
5.一种检测患者是否感染结核分支杆菌的方法，其特征在于：所述方法包括对炎症因子的检测，所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合，优选所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)中的一种或两种以上因子的组合。
6.根据权利要求5所述的用于检测患者是否感染结核分支杆菌的方法，其特征在于：所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A；所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ；所述肿瘤坏死因子选自：TNF、TNFα、TNFβ。
7.一种炎症因子在制备检测患者结核杆菌感染的诊断试剂盒中的用途，其特征在于：所述炎症因子选自细胞因子、C反应蛋白、白细胞计数中的一种或两种以上因子的结合，优选，所述细胞因子选自白细胞介素(Interleukin,IL)、干扰素(Interferon,IFN)、肿瘤坏死因子(Tumor-Necrosis Factor,TNF)中的一种或两种以上因子的组合。
8.根据权利要求7所述的炎症因子在制备检测患者结核杆菌感染的诊断试剂盒中的用途，其特征在于：所述白细胞介素选自：IL-1α、IL-1β、IL-4、IL10、IL11、IL12A；所述干扰素选自：IFNA1、IFNA2、IFNA3、IFNA4、IFNB、IFN-γ；所述肿瘤坏死因子选自：TNF、TNFα、TNFβ。
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