快速检测结核分枝杆菌异烟肼和利福平耐药相关基因mPCR-SSCP建立及应用

目的 建立快速检测结核分枝杆菌异烟肼(INH)和利福平(RFP)耐药相关基因katG、inhA和rpoB突变的多重聚合酶链反应-单链构象多态性(multi PCR-single strand conformational polymorphism analysis,mPCR-SSCP)方法.方法 药敏试验检测134株结核分枝杆菌临床菌株对INH和RFP的耐药性.设计结核分枝杆菌INH和RFP耐药相关katG、inhA和rpoB基因PCR引物,建立mPCR-SSCP技术检测上述菌株katG、inhA和rpoB基因的突变,同时采用PCR直接测序技术(PCR-DS)检测上述基因片段突变情况,并对上述3种方法检测结果进行分析和比较.结果 134株临床菌株均含有katG、inhA和rpoB基因,其中42株(31.3％)对INH耐药、45株(33.6％)对RFP耐药.mPCR-SSCP和PCR-DS检测结果显示,92株INH敏感菌株katG和inhA基因均未发生突变,检测特异性均为100％;89株RFP敏感菌株中rpoB基因分别有2株和1株检测出突变,检测特异性分别为97.8％和98.9％;42株INH耐药菌株中分别有33株和36株katG和/或inhA基因突变,检测灵敏度分别为78.6％和85.7％;45株RFP耐药菌株中rpoB基因分别有41株和43株发生突变,检测灵敏度分别为91.1％和95.6％.结论 本研究建立的mPCR-SSCP能快速、简便、特异,并有一定的敏感性检测结核分枝杆菌异烟肼和利福平耐药相关基因katG、inhA和rpoB突变,具有临床应用前景.     

显色法芯片检测结核分枝杆菌利福平和异烟肼耐药基因

目的建立显色法芯片检测结核分枝杆菌耐药基因的方法。方法设计4对地高辛标记引物,扩增结核分枝杆菌rpoB、katG、inhA和ahpC 4个基因部分片段,根据结核分枝杆菌利福平(RFP)和异烟肼(INH)4条耐药相关基因上8个位点的25种单核苷酸多态性设计探针制作芯片,扩增产物与芯片杂交,显色法判断结果;用该法检测46株结核分枝杆菌临床分离株。结果扩增产物琼脂糖电泳可见4条大小分别为165、181、245、315 bp DNA条带;结核分枝杆菌菌液浓度为1．6×103／ml时,该法仍可检测到各位点野生及突变信号;19种非结核分枝杆菌标准株和9种非分枝杆菌标准株扩增产物无DNA条带,与芯片杂交亦无信号,H37Rv结核分枝杆菌标准株芯片检测各位点均为野生型;5株结核分枝杆菌临床分离株重复检测5次,结果完全一致;6份PCR产物的测序结果与芯片检测结果完全一致;46株结核分枝杆菌临床分离株中,RFP耐药株28株,芯片检出突变株24株,突变率为85．7％,RFP敏感株18株,芯片检出突变株2株。INH耐药株31株,芯片检出突变株20株,突变率为64．5％,INH敏感株15株,芯片检出突变株4株。结论显色法芯片检测结核分枝杆菌耐药基因具有较高的敏感性和特异性,无需特殊仪器设备,有一定的推广应用价值。     

结核分支杆菌耐药基因检测对获得性耐药性的研究

了解不同年龄段肺结核病人的结核分支杆菌获得性耐药情况, 比较两种耐药性检测方法的检测效果, 评价它们的临床应用价值.用药敏试验(绝对浓度法)检测了结核分支杆菌株对RFP、INH、SM、PZA和EMB耐药情况, 采用聚合酶链反应-单链构象多态性分析(PCR-SSCP)检测结核分支杆菌耐药rpoB、katG、 rpsL、pncA和embB基因突变, 用绝对浓度法作药物敏感试验.青年组、中年组和老年组获得性耐药率分别为89.2%、85.3%和67.6%, 耐药基因突变率分别为76.2%、81.3%和63.2%,青年组耐药率和耐多药率显著高于其他组.结核杆菌耐药基因突变与耐药水平联系密切, 多数结核分支杆菌耐药基因突变易发生在高耐药区,也有少数基因突变发生在低耐药区.不规律用药时间越长耐药率越高.不同年龄组获得性耐药情况有差别, 应重视不同年龄组, 特别是青年组耐药率检测.PCR-SSCP是一种快速检测结核杆菌rpoB、katG、rpsL、pncA和embB基因突变敏感、特异的方法.     

恒温扩增检测结核分枝杆菌特异性IS6110片段在肺结核检测中的应用

目的 比较恒温扩增技术检测、抗酸杆菌涂片、结核分枝杆菌培养在肺结核患者检测中的应用.方法 收集2016年1月1日至12月31日我院呼吸科肺结核患者清晨痰液,将标本分为3份,分别进行痰涂片抗酸染色,结核菌培养及恒温扩增检测结核分枝杆菌特异性IS6110片段,统计分析3种方法的检测结果.结果 56例痰标本,恒温扩增检测结核分枝杆菌特异性IS6110片段阳性33例,阳性率为58.93％,抗酸染色阳性15例,阳性率为26.79％,恒温扩增检测结核分枝杆菌特异性IS6110片段阳性率高于抗酸染色方法;结核分枝杆菌培养阳性为35例,阳性率62.50％;恒温扩增检测结核分枝杆菌特异性IS6110片段和结核分枝杆菌培养两种方法检测Kappa系数为0.776,检测一致性较好.结论 恒温扩增检测结核分枝杆菌特异性IS6110片段是一种简便、高灵敏度的检出结核分枝杆菌的方法.     

荧光定量PCR检测病理组织中结核分枝杆菌的临床价值

目的探讨应用荧光定量PCR（FQ-PCR）技术在病理组织中检测结核分枝杆菌DNA（TB-DNA）的临床应用价值。方法对196例临床诊断结核的穿刺病理活检标本进行石蜡包埋切片,采用实时荧光定量PCR技术检测TB．DNA,并行抗酸染色及组织病理学检查,对三种检测与临床诊断符合率进行比较。结果在196例临床诊断结核的石蜡包埋组织中,荧光定量PCR检测阳性136例,阳性率为69．39％,抗酸染色检测阳性69例,阳性率为35．20％,组织病理学检查阳性102例,阳性率为52．04％,荧光定量PCR检测阳性率最高。结论荧光定量PCR技术简便、快捷,敏感性强、特异性高,可作为结核病分子病理诊断的重要检测方法,具有较高的临床应用价值。     

结核杆菌异烟肼耐药与katG基因突变分子的关系

近年来,全球结核病疫情回升,结核病依然是一个全球性的、严重的公共卫生问题和社会问题.异烟肼(isoniazid,INH)是治疗结核病的主要一线药物之一,2000年第4次结核病流行调查资料显示,INH的耐药率达17.6%,仍居第一位.故结核分枝杆菌对INH耐药的问题备受关注.新疆是我国耐多药结核病(MDR-TB)的高发区,研究新疆结核杆菌耐INH的机制,建立一种对临床分离株耐药的快速检测手段,是临床迫切需要解决的问题.     

应用反向线性杂交技术快速检测结核分枝杆菌利福平耐药性研究

探讨反向线性杂交技术(reverse line blot assay,RLB)在结核分枝杆菌利福平(RFP)耐药性快速检测中的应用价值.采用RLB将包含rpoB基因核心区的扩增产物与标记特异性探针的膜进行杂交,共对121株结核分枝杆菌进行RFP耐药性检测,并将结果与常规药敏实验进行比较.结果发现,121株中有71株对RFP耐药,56株为多重耐药株;采用RLB共检测到65株RFP耐药株rpoB基因核心区存在突变,其灵敏度为91.5%(65/71);50株RFP敏感株中均未检测到突变,则特异性为100%(51/51);56株多耐药菌株中,92.9%(52/56)存在rpoB基因核心区突变.因此,用RLB检测结核分枝杆菌RFP耐药株具有快速,高效,特异性和灵敏度高的优点,且可用于多耐药菌株的筛选,具有推广和潜在的临床应用价值.     

qPCR检测结核杆菌在肺和胸膜上皮样肉芽肿性疾病诊断中的意义

目的探讨荧光定量聚合酶链反应(Real-time polymerase chain reaction,q PCR)法在诊断及鉴别诊断肺和胸膜结核病和其他肉芽肿性疾病中的意义。方法收集经病理学检查发现上皮样肉芽肿性病变的肺和胸膜活检的石蜡包埋组织142例,应用q PCR法检测结核分枝杆菌DNA,同时行抗酸染色,并与临床资料对比进行回顾性分析。结果在142例肺胸膜活检标本中,60例临床诊断确诊为结核病。采用q PCR法检测结核分枝杆菌DNA阳性者58例,敏感性为96.67%,特异性为100%;抗酸染色阳性者17例,敏感性为28.33%,特异性为97.56%,差异有统计学意义(χ~2=21.47,P0.001)。结论结核分枝杆菌核酸检测有助于在石蜡包埋的肺和胸膜小活检组织中快速、准确的诊断结核病,可用于疑似结核的肉芽肿性病变的鉴别诊断。     

甲醛固定石蜡包埋切片经特殊染色后荧光定量聚合酶链反应检测的再利用

结核病是由结核分枝杆菌(mycobacterium tuberculosis, MTB)感染引起的特异性炎症,在甲醛固定石蜡包埋组织切片中查见MTB是确诊结核的"金标准".目前,结核病诊断的主要辅助检测方法是特殊染色和qRT-PCR检测.在临床病理诊断中,部分结核病患者由于获取标本困难,穿刺标本微小,病理组织多被用于H...     

EDTA修复肉芽肿组织中荧光定量PCR检测结核杆菌的应用

目的观察EDTA热修复炎性肉芽肿组织石蜡切片,能否提高荧光定量PCR结核/非结核分枝杆菌的检出率。方法对125例炎性肉芽肿组织石蜡切片结核/非结核分枝杆菌同时进行常规荧光定量PCR检测和EDTA热修复后荧光定量PCR检测。结果常规荧光定量PCR检测检出分枝杆菌75例(60%)阳性,其中结核杆菌74例(59.2%),非结核分枝杆菌1例(0.8%),阳性病例平均阳性基因拷贝数6.35×105/ml/例;EDTA修复后荧光定量PCR检测检出分枝杆菌88例(70.4%),其中结核杆菌83例(66.4%),非结核分枝杆菌5例(4%),阳性病例平均阳性基因拷贝数7.36×106/ml/例。两组间差异均有统计学意义(P0.05,P0.01)。结论 EDTA热修复后荧光定量PCR检测可以大幅度提高炎性肉芽肿组织石蜡切片的结核/非结核分枝杆菌的阳性检出率。     

Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.     

HRCA技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变的初步分析

目的 对超分支滚环扩增技术(hyperbranched rolling cycle amplification, HRCA)技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变进行初步分析.方法 采用直接涂片检测在2013年至2015年期间搜集的898份痰液样本中结核分枝杆菌情况,同时采用改良罗氏培养法与HRCA技术联合DNA芯片法对阳性样本进行鉴定,并对利福平耐药基因进行初步分析.结果 989份检测的痰液样本中361份为阳性,涂阳率为40.2%.HRCA技术联合DNA芯片法阳性率为98.0%,显著高于改良罗氏培养法(35.2%),且差异具有统计学意义(P<0.05).通过HRCA技术联合DNA芯片法发现85株结核分枝杆菌对利福平耐药,其中单纯rpoB 基因突变比例为36.5%(31/344);katG基因与rpoB基因均突变比例为60.0%(51/344);inhA基因与rpoB基因突变比例均为3.5%(3/344);且二位点联合突变及单位点突变率分别为89.4%(76/85)与10.6%(9/85).结论 HRCA技术联合DNA芯片法能够有效快速检出结核分枝杆菌突变基因,rpoB基因突变为主要结核分枝杆菌利福平耐药的基础,且突变呈现多样性.     

Sensitive differential detection of genetically related mycobacterial pathogens in archival material

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.     

A combination of two genetic markers is sufficient for restriction fragment length polymorphism typing of Mycobacterium tuberculosis complex in areas with a high incidence of tuberculosis

The incidence of tuberculosis (TB) in Madagascar is 150 cases per 100,000 people. Because of this endemicity, we studied the genetic diversity of Mycobacterium tuberculosis strains isolated in four big cities in 1994 to 1995 with the aim of monitoring TB transmission. Isolates from 316 cases of pulmonary TB (PTM(+)) were typed by Southern hybridization with genetic markers IS6110 and DR. Of the 316 PTM(+) strains, 66 (20.8%) had a single IS6110 band and were differentiated by the DR marker into 33 profiles. Using both markers, 37.7% (119) of the patients were clustered, a proportion similar to that in countries with a high prevalence of TB. There was no significant difference between clustered and nonclustered patients in age, sex, Mycobacterium bovis BCG status, and drug susceptibility of strains. Clustering was significantly greater in the capital, Antananarivo, than in the other cities, suggesting a higher rate of transmission. However, most of the patients in clusters were living in different areas, and, within a distance of 0.7 km, we did not find epidemiologically unrelated strains with the same restriction fragment length polymorphism profile. Despite an apparently low polymorphism, genetic markers such as IS6110 are potentially valuable for monitoring TB transmission. However, the high proportion of Malagasy isolates with a single IS6110 copy makes this marker alone unsuitable for typing. Additional markers such as DR are necessary for the differentiation of the isolates and for epidemiological surveys.     

Improved Case Detection Using Xpert Mycobacterium tuberculosis/Rifampicin Assay in Skeletal Tuberculosis

Background: In India, musculoskeletal tuberculosis (TB) accounts for 10%–25% of extrapulmonary TB. Data on drug-resistant skeletal TB are lacking. At present, the diagnosis is based mainly on radiological techniques. Laboratory confirmation of skeletal TB is delayed as 6–8 weeks are required for culture results. Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay is a fully automated test which simultaneously detects MTB and RIF resistance within 3 h. Hence, this study was done to compare the yield of case detection using Xpert assay in comparison with culture in specimens received from clinically suspected skeletal TB cases. Methods: Retrospective analysis of microscopy, culture and Xpert assay results was carried out on specimens received in laboratory from skeletal TB cases from January 2016 to December 2017. Results: Of the 201 patients analysed, majority of the specimens were obtained from the spine (55.72%). MTB was detected in 48.68% of tissue and 24% of pus specimens. Xpert assay was detected MTB in 67 (33.33%) specimens of which 53 (47.32%) were from the spine. Culture was detected MTB in 66 (32.83%) specimens. Xpert assay was detected two specimens more than culture. One specimen was positive by only culture. RIF-resistant MTB was detected in 10 (14.92%) specimens by Xpert assay. Conclusion: The spine is the most common site involved. Tissue specimen is better for early diagnosis. High RIF resistance in skeletal TB is an alarming situation. Ability of Xpert MTB/RIF assay for rapid and simultaneous detection of MTB and RIF resistance in comparison with culture makes it a useful diagnostic tool in skeletal TB.     

Performance of Xpert MTB/RIF RUO assay and IS6110 real-time PCR for Mycobacterium tuberculosis detection in clinical samples

The Cepheid Xpert MTB/RIF research-use-only (RUO) assay and a laboratory-developed test (LDT) targeting IS6110 were evaluated and compared to mycobacterial culture as the gold standard. The performance characteristics of both molecular assays were determined by using 112 specimens from 90 patients, including 89 pulmonary specimens and 23 extrapulmonary specimens. Of the specimens tested, 37 (33%) were culture positive for the Mycobacterium tuberculosis complex; 29 were pulmonary, and 8 were extrapulmonary. Of these culture-positive specimens, 83% of the pulmonary specimens and 50% of the extrapulmonary specimens were smear positive. There was complete concordance between the smear-positive culture-positive specimens, independent of the anatomical site (100% sensitivity). The sensitivity of the MTB/RIF RUO assay for smear-negative specimens was 60% for pulmonary and 75% for extrapulmonary specimens, while the IS6110 LDT sensitivities were 40% and 0%, respectively. There was also complete concordance among the culture-negative specimens tested. Both assays showed 95% specificity, with four culture-negative specimens testing as positive. A review of patient records indicated that there was a high likelihood of the presence of M. tuberculosis complex DNA in the false-positive specimens. Biosafety analysis was performed and showed an acceptable reduction in organism viability using the processing methods described above. Both molecular assays are suitable for the detection of M. tuberculosis isolates in smear-positive pulmonary and extrapulmonary specimens, while the sensitivity of the detection of M. tuberculosis isolates in smear-negative specimens was variable.     

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.     

Spread of drug-resistant pulmonary tuberculosis in Estonia

Restriction fragment length polymorphism (RFLP) analysis of 209 Mycobacterium tuberculosis clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS6110 banding pattern. These strains shared the majority of their IS6110 DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%). This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains. The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of M. tuberculosis belonged to family A. These strains were confirmed by spoligotyping as members of the Beijing genotype family. The spread of Beijing genotype MDR M. tuberculosis strains was also frequently seen in 1997 to 1999. The members of this homogenous group of drug-resistant M. tuberculosis strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.     

分析5种结核杆菌耐药基因突变与耐药水平的关系

目的：分析5种结核杆菌（M.tb)耐药基因突变的情况，了解基因突变和耐药水平的关系。方法：134例临床分离株均做传统梯度药敏试验和聚合酶链反应－单链构象多态性I（PCR－SSCP）试验。结果：耐PZA(pncA),SM(rpsL),REP(rpoB),INH(katG),EMB(embB)基因突变率分别为42．7％、72％、78％、69％和43．9％，其中，上述高耐株基因突变率分别为70％、87．2％、93．4％、80％、43．9％。低耐株分别为12．5％、28．5％、45．4％、18．7％，EMB在低耐区无基因突变，结论：M.tb耐药基因变与耐药水平联系密切，多数M.tb耐药基因突变易发生在高耐药区，也有少数菌基因突变易发生在低耐药区。