结核分枝杆菌抗体检测蛋白芯片的制备及应用

目的:用重组结核分枝杆菌ESAT6、CFP10、M16和M38抗原制备相应的抗体检测蛋白芯片。方法:将制备的结核分枝杆菌抗原ESAT6、CFP10、M16和M38及购买的LAM抗原点于醛基化修饰的玻片上,制备成结核抗体检测蛋白芯片;使用该芯片对130例临床结核病患者和50例健康体检者血液样品进行检测,分析其敏感性和特异性,以及5项结核抗体的构成比。结果:结核杆菌抗体检测蛋白芯片的敏感性为90.8%(118/130),特异性为90%(45/50),LAM的检出率最高为91.5%。结论:用ESAT6、CFP10、M16和M38及LAM抗原制备的结核杆菌抗体检测蛋白芯片用于结核病辅助诊断的敏感性和特异性较高,可用于结核分枝杆菌抗体检测蛋白芯片试剂盒的开发。     

结核杆菌可溶性细胞壁抗原与重组38Kd蛋白用于结核病血清学诊断的价值

目的探讨结核杆菌CW抗原和rTPA38蛋白用于结核病血清学诊断的价值。方法以CW和rTPA38蛋白为抗原,LAM为对照,用DICFA检测血清中的抗结核抗体。结果191例肺结核病人血清,用CW、rTPA38和LAM检测的敏感性分别为78．0％、65．5％和72．3％,特异性分别为95．9％、98．4％和95．9％。统计分析显示CW和rTPA38检测肺结核病人血清抗结核抗体的敏感性差异有非常显著性（χ^2=16．230,P〈0．01）。两者检测健康人和非结核组病人血清的特异性差异有显著性（χ^2=3．972,P〈0．05）。检测痰涂片阳性血清86例,发现CW和rTPA38与痰阳的一致率分别为84．9％和69．8％,CW抗原与痰涂片的阳性反应明显高于rTPA38。结论CW抗原有较好的敏感性和特异性,且与痰涂片有较高的符合率,有助于结核病的血清学诊断。     

结核病血清学诊断进展

结核病的细菌学检查目前是结核病实验室诊断的金标准.由于痰涂片阳性率较低,镜检需要经验,难以区分环境分枝杆菌造成的假阳性;痰培养需时太长,因此细菌学检查对结核病的诊断价值有限.随着分子生物学技术的迅速发展,用高度敏感的方法检测结核分枝杆菌(简称结核杆菌)及其特异性DNA片段,如聚合酶链反应(PCR)、生物探针和基因芯片等,需要相应的检测设备和检测费用高而未能广泛推广.血清学诊断技术有其固有的优势,如操作简便、快速、便于推广、无需特殊精密仪器,已有多种纯化的特异性抗原可采用,可以发展成自动化检测,尤其是对痰培养阴性和肺外结核病有较为重要的辅助诊断价值而受到广泛的重视.本文从靶抗原的选择和检测技术的改进等方面综述了结核病血清学诊断的现状和进展.     

结核分枝杆菌CysA2抗原的基因克隆、表达及在血清诊断中的应用

目的 克隆并构建结核分枝杆菌CysA2基因原核表达系统,建立基于rCysA2的ELISA并检测rCys A2-ELISA在肺结核患者血清学诊断中的敏感性和特异性.方法 采用PCR扩增CysA2基因,构建CysA2基因原核表达系统.采用SDS-PAGE检测目的重组蛋白rCysA2表达情况,Ni-NTA亲和层析法提纯rCysA2,免疫印迹鉴定rCysA2的免疫反应性.以rCysA2为包被抗原,建立rCysA2-ELISA并用于检测132例血清抗体,其中涂片阳性和阴性肺结核患者血清分别占96例和36例,非肺结核肺部疾病病人血清抗体30例,健康对照血清50例.结果 克隆的CysA2基因与GenBank中相关序列相似性为100％.rCysA2表达量为细菌总蛋白的25.4％.提纯的rCysA2在SDS-PAGE后显示为单一的蛋白条带.rCysA2-ELISA检测涂片阳性和涂片阴性的肺结核患者血清抗体检出率分别为63.5％和61.1％,50例健康对照全部阴性、30例非肺结核肺部疾病患者1例呈现阳性,此方法的灵敏度为62.9％(83/132),特异性为98.8％ (79/80).结论 本研究成功地克隆并构建了结核分枝杆菌CysA2基因及其原核表达系统.以rCysA2为包被抗原的rCysA2-ELISA方法有较高的灵敏度和很好的特异性,能有效检测涂片阴性患者血清抗体,rCysA2有望成为结核病血清诊断的有效候选抗原之一.     

结核分枝杆菌Rv3425特异性蛋白片段与CFP10-ESAT6融合蛋白在结核抗体检测中的应用价值

目的:对结核分枝杆菌Rv3425蛋白进行生物信息学分析及抗原表位预测,评价Rv342519-176重组蛋白与CFP10-ESAT6融合蛋白在结核抗体检测中的应用。方法:PCR扩增结核杆菌Rv3425蛋白19～176位的编码基因片段,原核表达并纯化Rv342519-176重组蛋白(rRv342519-176)和CFP10-ESAT6融合蛋白(rCFP10-ESAT6),建立以重组蛋白为抗原的ELISA方法,评价2种蛋白在结核抗体检测中的联合应用价值。结果:具有抗原表位的rRv342519-176与rCFP10-ESAT6在大肠杆菌中高效表达;ELISA结果显示,rRv342519-176的敏感性为39%,特异性为97.5%;rCFP10-ESAT6的敏感性为37%,特异性为95%;2种蛋白联合检测,敏感性提高到57%,特异性为95%。结论:生物信息学预测Rv3425的优势抗原表位,表达并纯化的rRv342519-176和rCFP10-ESAT6在结核抗体检测中具有一定的抗原互补性,在保持特异性的基础上可显著提高检测敏感性。     

结核分枝杆菌Rv3871抗原优势肽段的克隆表达及抗原性鉴定

目的:为了提高结核病诊断试剂的特异性和敏感性,克隆表达结核分枝杆菌H37Rv株RD1区Rv3871抗原优势肽段,并应用ELISA法对其抗原性进行初步鉴定。方法:利用Biosun生物信息学软件对Rv3871抗原进行表位分析,通过PCR从结核分枝杆菌H37Rv基因组中扩增Rv3871抗原优势肽段编码基因,在大肠杆菌HB101中进行表达,采用间接ELISA法初步评价其抗原性。结果:Rv3871-1肽段检测的敏感性为32.5%(13/40),特异性为97.2%(35/36);Rv3871-2敏感性为45%(18/40),特异性为100%;Rv3871-3敏感性为37.5%(15/40),特异性为91.6%。结论:结核分枝杆菌RD1区Rv3871抗原优势肽段Rv3871-2有较高的特异性和敏感性,有望作为候选抗原用于结核病患者的血清学检测。     

结核分枝杆菌抗原检测研究进展

结核病仍然是一个严重的全球性公共卫生问题,有效控制结核病的障碍在于缺乏早期、准确的诊断方法。机体受到结核分枝杆菌感染后,体内首先出现的是结核分枝杆菌特异性抗原。因此,结核分枝杆菌抗原检测作为结核病早期诊断的方法可能具有很高的诊断价值。我们简要综述了结核分枝杆菌抗原检测的相关研究进展。     

结核分枝杆菌抗体双抗原夹心ELISA检测方法的建立及初步应用

目的:建立2种结核分枝杆菌(Mtb)抗体的双抗原夹心ELISA检测方法,并初步评价其在Mtb血清学临床辅助诊断中的应用价值。方法:采用Mtb融合抗原38k D+ESAT6+CFP10作为包被抗原,分别以辣根过氧化物酶(HRP)和生物素(Bio)标记的38k D+ESAT6+CFP10作为标记抗原,建立2种Mtb双抗原夹心ELISA法,即HRP-ELISA法和Bio-ELISA法;采用所建立的2种方法对结核患者和健康对照血清进行检测。结果:经过一系列的反应条件优化,确定HRP-ELISA法和Bio-ELISA法的最佳抗原包被浓度分别为2、0.25μg/m L,最适加样量均为100μL血清原液,最佳标记抗原稀释率分别为1∶500、1∶2000,检测的灵敏度分别为43.66%、52.11%,特异性均为100%。结论:建立了2种Mtb双抗原夹心ELISA法,它们均适用于Mtb血清学的临床辅助诊断。     

结核分枝杆菌Rv3881c突变子的克隆及其在大肠杆菌中的高效表达

目的：融合表达结核分枝杆菌Rv3881c突变子抗原,以便与其他已知的免疫显性抗原联合使用,有效增加结核分枝杆菌感染血清学检验的敏感性和特异性。方法：将克隆的来源干结核分枝杆菌H37Rv株的Rv3881c突变体基因插入原核表达载体pET24b,并在大肠杆菌BL21中获得高效表达;由于重组的Rv3881c突变子抗原片段同C端的6xHis标签融合表达,使用Ni-柱进行快速纯化。结果：Western印迹表明,重组的Rv3881c突变子抗原同选取的6例结核病阳性临床血清标本均能发生明显的反应。结论：重组Rv3881c突变体具有较好的特异性,提示该抗原可能成为检测结核的有效抗原之一。     

检测脂阿拉伯甘露聚糖诊断结核病

结核病的诊断是靠直接痰涂片抗酸染色和培养。然而,直接涂片的敏感性仅22～43％,培养虽比直接痰涂片法敏感,但又耗时较长,而且只限于具备培养条件的实验室。 结核病的快速诊断方法主要有两类:一是检测结核糖菌的抗原,如结核性脑膜炎的诊断;二是采用探针技术检测结核杆菌的核酸。采用聚合酶链反应(PCR)技术诊断结核病确属一种理想的方法,     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Diagnostic Accuracy of the PURE-LAMP Test for Pulmonary Tuberculosis at the County-Level Laboratory in China

Background

Early and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China.

Methodology/Principal Findings

From April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture.

Conclusions/Significance

The study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.     

Expression and purification of recombinant 38-kDa and Mtb81 antigens of Mycobacterium tuberculosis for application in serodiagnosis

Availability of genome sequence of Mycobacterium tuberculosis has accelerated identification of antigens for serodiagnosis of tuberculosis and a number of new antigens are being tested in various combinations to produce cocktails with high sensitivity and specificity. For producing a highly specific diagnostic test, it is important that the recombinant antigens be highly pure, free of host protein, and correctly folded so that they bind only to specific antibodies. Also, for commercial viability they need to be produced in high yields. We have cloned, expressed, and purified a number of mycobacterial antigens in Escherichia coli. This paper describes, expression and purification of two important mycobacterial proteins with serodiagnostic potential, namely, 38-kDa and Mtb81 antigens, in monomeric form. The protocol involves using a T7 promoter based expression vector under conditions of regulated and slow expression followed by three-step column chromatography procedure to obtain highly purified proteins. The yields of the two proteins were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of tuberculosis patients (smear positive, smear negative, and extra-pulmonary categories).     

Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis

Background

Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples.

Methodology/Principal Findings

In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as Löwenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively.

Conclusions/Significance

The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient.     

Xpert MTB/RIF在人类免疫缺陷病毒感染/艾滋病患者中诊断结核病的价值

为探讨利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert Mycobacterium tuberculosis/rifampicin,Xpert MTB/RIF)在人类免疫缺陷病毒感染/艾滋病(human immunodeficiency virus infection/acquired immunodeficiency syndrome,HIV/AIDS)患者中诊断结核病的价值,本研究回顾性分析了2018年1月1日—2020年12月31日复旦大学附属公共卫生临床中心感染与免疫科收治的801例HIV/AIDS合并疑似结核病患者的临床资料。801例患者中,657例进行了Xpert MTB/RIF、外周血结核感染T细胞斑点试验(tuberculosis T cell spot test,T-SPOT.TB)、抗酸染色涂片镜检和BACTEC MGIT 960液体培养等检测。以液体培养及菌型鉴定结果作为结核病诊断的“金标准”,确诊结核病92例,Xpert MTB/RIF、T-SPOT.TB、抗酸染色涂片镜检在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)的灵敏度分别为72.8%、55.4%和69.6%,特异度分别为96.8%、90.3%和84.4%,与“金标准”行一致性检验,Kappa值分别为0.719 (P<0.01)、0.430(P<0.01)和0.424(P<0.01)。Xpert MTB/RIF检测502份呼吸道样本,结果显示其诊断肺结核的灵敏度和特异度分别为66.7%和96.0%;在痰涂片阳性和阴性的患者中,Xpert MTB/RIF诊断肺结核的灵敏度分别为77.4%和35.2%,特异度分别为87.7%和 97.8%。采用Xpert MTB/RIF检测343份肺外标本,结果显示其诊断肺外结核的灵敏度和特异度分别为63.3%和95.2%。以上结果提示,Xpert MTB/RIF在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)具有较高的灵敏度和特异度,诊断肺结核的灵敏度高于肺外结核,因此推荐将其作为HIV/AIDS患者疑似结核病的首选检测方法。     

Expression and purification of recombinant antigens of Mycobacterium tuberculosis for application in serodiagnosis

Accurate diagnosis is essential for the treatment, prevention, and control of tuberculosis. Poor specificity of the tuberculin skin test in BCG-vaccinated populations and constraints to implementation of PCR and CMI-based diagnostic assays in developing countries warrant development of easy-to perform robust serological tests. Due to great heterogeneity in humoral response in TB patients, it will be necessary to include several antigens in any diagnostic assay to achieve useful levels of sensitivity and specificity. This needs production of recombinants, soluble versions of mycobacterial antigens in high yields. We have cloned, expressed, and purified a number of mycobacterial proteins in Escherichia coli. This paper describes the expression and purification of four promising sero-reactive proteins namely, ESAT6, CFP10, MTC28, and 14-kDa antigen of Mycobacterium tuberculosis. The protocol involves regulated and slow expression of proteins by using a T7 promoter-based expression vector for obtaining soluble protein followed by a three-step column chromatography procedure employing media with high binding capacity and flow characteristics. The yields of these proteins obtained were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of TB patients (smear positive, smear negative, and extra-pulmonary categories) and in combination with other immunodominant antigens will be useful in increasing the sensitivity to detect M. tuberculosis specific antibodies.     

Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

上海口岸2014-2015年入境人员肺结核筛查情况

为了解上海口岸入境人员肺结核的筛查情况及后续处理,防止结核病通过口岸跨境传播,本研究于2014年1月—2015年12月对所有在上海口岸办理入境体检的14岁以上人员进行结核病筛查,通过病史、体格检查和胸部X线摄影筛查疑似肺结核患者;对疑似肺结核患者进行痰细菌学检测、T‐SPOT ．TB和 Xpert MTB／RIF检测。结果显示,2014—2015年上海口岸入境人员共检出疑似肺结核患者215例,总检出率为229．76／10万;确诊肺结核患者33例,总检出率为35．27／10万,确诊率为15．3％。对210例疑似肺结核患者进行痰细菌学检测,结果显示结核分枝杆菌培阳率为14．3％,非结核分枝杆菌培阳率为17．1％。有95例和78例疑似肺结核患者分别接受 T‐SPOT ．TB和 Xpert MTB／RIF 检测,以痰细菌学检测为“金标准”,T‐SPOT ．TB的灵敏度为100％,特异度为49．4％;Xpert MTB／RIF的灵敏度为87．5％,特异度为96．8％。33例确诊肺结核患者中,25例（75．8％）离境,15例（45．5％）在离境前接受抗结核治疗,8例（24．2％）失访。本研究显示,上海口岸入境人员中肺结核确诊率仍有待提高。筛查与诊断中,T‐SPOT ．TB具备较高灵敏度, Xpert MTB／RIF具备较高特异度,两种方法均有较高应用价值,两者联用可提高检出率,缩短检出时间。对确诊病例或未确诊的可疑病例应加强后续监管。     

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.