聚合酶链反应检测肺结核患者外周血单个核细胞中结核分支杆菌DNA的临床价值

应用聚合酶链反应（ＰＣＲ）技术配对检测９２例肺结核患者外周血单个核细胞内和痰标本中结核分支杆菌ＤＮＡ，同时检测３０例非结核患者外周血。结果显示：外周血和痰标本ＰＣＲ阳性率分别为７１７％和５５４％，前者明显高于后者（Ｐ＜００５）；各型肺结核外周血和痰标本ＰＣＲ阳性率不尽相同；３０例非结核患者外周血ＰＣＲ阳性３例（１０％）。认为ＰＣＲ检测肺结核患者外周血单个核细胞内结核分支杆菌ＤＮＡ是一种快速、敏感的方法，可用于肺结核早期诊断与鉴别诊断     

AmpliSensor—聚合酶链反应定量检测肺结核患者外周血结 …

目的 探讨ＡｍｐｌｉＳｅｎｓｏｒ－聚合酶链反应定量检测外周血中结核分支杆菌ＤＮＡ在肺结核的应用价值。方法 采用ＱｌＡａｍｐ和ＡｃｕＰｕｒｅ法提取，制备全血中模板ＴＢ－ＤＮＡ，应用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ定量检测，并与ＩＳ６１１０－单管巢式聚合酶链反应（ＳＮ－ＰＣＲ）作比较。结果２００例肺结核患者的血液标本中，两种方法测得结核分支杆菌ＤＮＡ的阳性率分别为６０．５％、６３．５％。８５例非结核肺病     

聚合酶链反应技术检测咽拭子标本中结核菌DNA的评价

为探讨聚合酶链反应（PCR）检测咽拭子标本中结核菌在诊断肺部疾病的价值。应用PCR法检测肺结核患者50例，非肺结核患者55例及100例正常健康人咽拭子标本中结核菌和咽拭子涂片找抗酸杆菌，并与痰涂片检查作比较。肺结核组、非肺结核组及正常健康人咽拭子PCR阳性率分别为60％、3O．9％及10％，涂片为6％、0％和0％。结核组及非结核组痰PCR阳，牲率为54％和10．9％，涂片为26％和0％。结果表明PCR检测咽拭子标本结核菌DNA是肺结核病早期诊断和鉴别诊断的一种有价值的检测手段。但应注意假阳性或假阴性出现的可能。     

巢式聚合酶链反应检测支气管内膜结核患者活检组织中结核 …

目的 了解巢式聚合酶链反应（ＮＰＣＲ）技术对支气管内膜结核的诊断价值。方法 应用巢式聚合酶链反应（ＮＰＣＲ）对６７份支气管内膜活检组织进行结核分支杆菌ＤＮＡ检测。并与病理检查,刷检涂片,支气管镜检后痰涂片和痰培养结果比较,对照为４３例支气管肺癌患者,结果６７例支气管内膜结核活检组织病理检查,刷检涂片,支气管镜检“激惹”后痰涂片,镜检术后痰培养及ＮＰＣＲ检测阳性率分别为１３％,１９％,２２％,１５％     

肺结核患者外周血白细胞中结核分支杆菌DNA检测及实验评价

目的 探讨结核菌检测的新途径及其在肺结核诊断中的价值。方法 应用聚合酶链反应技术对７６例肺结核患者（４０例同时留有痰标本），４２例非结核患者外周血标本进行了结核分支杆菌ＤＮＡ检测。结果 利用聚合酶链反应检测外周血白细胞中结核菌ＤＮＡ对肺结核诊断的灵敏度为８６％，特异性为９０％，准确度达８７％；４０例外周血与痰标本的ＰＣＲ配对检测表明，血标本中的结核菌ＤＮＡ检出率明显高于痰标本。结论 外周血细胞中结     

聚合酶链反应检测结核分支杆菌临床标本制备方法的研究

分别检测６种结核菌ＤＮＡ提取方法的裂解效能、聚合酶链反应（ＰＣＲ）扩增灵敏度及其对临床标本的阳性检出率。结果显示，ＴＥ－ＴｒｉｔｏｎＸ法和Ｃｈｅｌｅｘ－１００法能裂解绝大多数的细菌并具最高的敏感性，可检测出少至１０个菌细胞，其临床标本的阳性检出率分别为９３．３％和８６．６％；而其余４种方法效果较差，ＴＥ－Ｔｒｉｔｏｎ和Ｃｈｅｌｅｘ－１００两种沸水浴法简捷、经济、高效，值得临床实验室推广使用。     

痰结核杆菌DNA的聚合酶链反应检测及应用

痰结核杆菌ＤＮＡ的聚合酶链反应检测及应用陈良玺，宋世云，赵秀菊，吕云生，季学东陈光连，邢桂云，管庆兰，马振霞，付勋杰我们对１８８例结核病患者进行聚合酶链反应（ＰＣＲ）检测，并同时与直接涂片镜检法、培养检查结核菌和非结核患者作对照进行了分析研究，现报告...     

聚合酶链反应DNA扩增对活动性肺结核的诊断价值

为探讨聚合酶链反应（ＰＣＲ）ＤＮＡ扩增对诊断活动性肺结核的临床价值，本文用ＰＣＲ技术扩增结核杆菌特有的ＭＰＢ６４蛋白基因序列，对１２１例病人临床标本进行检测。发现３４例活动性肺结核中有２８例ＰＣＲ阳性；疑诊为肺结核者３３例中有１８例阳性；非结核肺疾患５４例中有１０例阳性，其中有肺结核既往史者阳性６例，有结核接触史者阳性３例。提示ＰＣＲ阳性并非为活动性肺结核所特有。     

结核分支杆菌DNA的单管巢式聚合酶链反应检测

目的探讨单管巢式聚合酶链反应（ＳＮＰＣＲ）检测石蜡包埋组织结核分支杆菌ＤＮＡ的特异性和敏感性。方法应用普通ＰＣＲ（ＧＰＣＲ）、双管巢式ＰＣＲ（ＤＮＰＣＲ）和ＳＮＰＣＲ对结核分支杆菌ＢＣＧ和３０例结核性淋巴结炎石蜡包埋组织进行结核分支杆菌复合群ＩＳ６１１０特异插入序列片段ＤＮＡ检测。结果ＤＮＰＣＲ和ＳＮＰＣＲ检测ＢＣＧＤＮＡ均于１５ｆｇ以上呈现阳性结果，其敏感性明显优于ＧＰＣＲ（４８０ｆｇ）。ＧＰＣＲ、ＤＮＰＣＲ和ＳＮＰＣＲ检测３０例结核性淋巴结炎阳性率分别为４３％、１００％和１００％，抗酸染色阳性率（１０％）与３种ＰＣＲ法相比差异有非常显著意义（均Ｐ＜０．０１）。ＧＰＣＲ阳性率与ＤＮＰＣＲ和ＳＮＰＣＲ相比差异亦具非常显著意义（均Ｐ＜０．０１）。ＳＮＰＣＲ阳性率与ＤＮＰＣＲ相同。结论巢式ＰＣＲ检测淋巴结石蜡包埋组织结核分支杆菌的敏感性显著高于ＧＰＣＲ，其中ＳＮＰＣＲ具有与ＤＮＰＣＲ相同的特异性和敏感性，并具有更大的实用价值。     

应用多重聚合酶链反应法检测并鉴别结核分支杆菌与非结核分支杆菌DNA

OBJECTIVE: To improve the specificity and sensitivity of polymerase chain reaction (PCR) technique for detecting and identification of DNA of M. tuberculosis and M. nontuberculosis. METHOD: Three pairs of oligonucleotide primer were used in triplex-PCR. A 383 bp DNA fragment encoding part of the 65,000 mycobacterial surface antigen, a 123 bp fragment corresponding to a specific M. tuberculosis complex sequence which was the insertion sequence 6110 (IS 6110) and a 268 bp fragment for human beta-globin were amplified by triplex-PCR respectively. RESULT: The sensitivity of the triplex-PCR-electrophoresis for the DNA of mycobacterium was 0.6 pg. The specific bands of 383 bp and 123 bp among the amplified DNA from M. hominis, M. bovis, BCG and M. simiae were present in the agarose gel. By contrast, only a band of 383 bp was found among the M. nontuberculosis which contained M. avium, M. chelonae, M. scrofulaceum, M. xenopi, M. kansasii, M. intracellulare and M. smegmatis. Compared with the standard strains, there was an additional 268 bp band in simulated clinical samples infected by Mycobacterium. The above 3 specific bands were found neither in other 15 bacterial species tested nor in Mycoplasma pneumoniae. 182 clinical samples were examined by culture, smear and triplex-PCR. 72 nontuberculous clinical samples were all negative. In 110 tuberculosis clinical samples, the positive rates were 2.7%, 13.6% and 32.7%, respectively. CONCLUSION: The triplex-PCR possesses a high specificity and sensitivity. This method could detect and identify the DNA of M. tuberculosis and M. nontuberculosis except M. simiae. It is a valuable tool for early diagnosis and differentiation for infection of M. tuberculosis and M. nontuberculosis.     

聚合酶链反应对菌阳肺结核治疗的监测

目的探讨痰菌阳性肺结核患者在治疗期及停药２年内痰结核分支杆菌及其ＤＮＡ阴转情况与复发的关系以及聚合酶链反应（ＰＣＲ）对菌阳肺结核患者治疗的监测价值。方法用ＰＣＲ技术、涂片及培养法对８７例菌阳肺结核于治疗期每月检测１次，停药期继续随访２年。结果痰结核分支杆菌ＰＣＲ转阴时间通常比涂片和培养迟１～３个月，痰含菌量越多，ＰＣＲ持续阳性时间越长。８７例中１０例（１１％）ＰＣＲ持续阳性１年以上，其中３例（３０％）分别于停药后８、１２、１６个月时复发，１例ＰＣＲ已转阴病例于１８个月时复发。此４例均有痰菌复阳，胸片示病灶增多而再次接受治疗。结论ＰＣＲ用于临床疗效观察比涂片、培养实用，对估计有可能复发的病例有一定帮助。     

AmpliSensor-聚合酶链反应技术检测结核分支杆菌及临床应用

目的探讨Ａｍｐｌｉｓｅｎｓｏｒ－聚合酶链反应（ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ）在结核病诊断中的价值。方法采用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ对７８４例结核病患者及１６０例肺癌患者的标本进行检测，并与ＰＣＲ（凝胶电泳后，经溴化乙锭染色）、涂片、培养等法比较。结果ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ的敏感性显著高于涂片及培养（Ｐ＜０．０１）。特异性较ＰＣＲ法高。结论ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ可以通过标准曲线划定检出下限，并可换算出标本中原始的靶ＤＮＡ值，同时具有较高的特异性和敏感性，对肺结核尤其是肺外结核的诊断有一定的临床意义。     

TaqMan聚合酶链反应技术检测结核分支杆菌DNA及其临？…

目的 探讨ＴａｑＭａｎ聚合酶链反应（ＴａｑＭａｎ－ＰＣＲ）技术在肺结构诊断中的价值。方法 对１６８例活动性肺结核、５７例肺癌患者的痰和外周血及３４－例健康对照外周血,同时应用ＴａｑＭａｎ－ＰＣＲ、ＰＣＲ检测,并与痰涂片法、ＢＡＣＴＥＣ法及改良罗氏培养法结果进行比较。结果 ＴａｑＭａｎ－ＰＣＲ检测痰和外周血总的阳性率分别为５３．０％和６１．３％,显著高于ＰＣＲ、痰涂片法、ＢＡＴＣＴＥＣ法及改良罗氏培     

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens ,

OBJECTIVE:

To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens.

METHODS:

We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results.

RESULTS:

Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively).

CONCLUSIONS:

Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.     

Detection of Mycobacterium tuberculosis in bronchoalveolar lavage from patients with sputum smear-negative pulmonary tuberculosis using a polymerase chain reaction assay

Abstract The objective of this study was to evaluate the utility of a polymerase chain reaction (PCR) assay in detecting Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) specimens of patients suspected of having active pulmonary tuberculosis (TB) but who were sputum smear-negative. Patients undergoing investigation for suspected pulmonary TB at the University Hospital, Kuala Lumpur, and who were sputum smear-negative underwent fibreoptic bronchoscopy and BAL. One portion of each lavage specimen was submitted for smear examination for acid-fast bacilli and mycobacterial culture and the other portion assayed by PCR for the presence of a 562-base pair DNA segment belonging to the insertion sequence IS986, unique to the M. tuberculosis complex. As controls, lavage specimens from patients with other lung lesions were also similarly tested. The PCR assay gave a positivity rate of 80.9% (55 of 68) compared with 8.8% of smear examination and 7.4% of culture for detecting M. tuberculosis in BAL specimens. The assay was positive in two of 45 BAL specimens from 35 control subjects. The PCR assay was more sensitive than smear and culture in detecting M. tuberculosis in BAL specimens of patients with sputum smear-negative pulmonary TB.     

实时定量聚合酶链反应技术在鉴别结节病与增殖性结核病中的应用

目的利用实时定量聚合酶链反应（PCR）方法检测结节病和结核病病理组织中结核分枝杆菌DNA，以探讨结核分枝杆菌在结节病发病中的作用及本方法在结节病与增殖性结核病鉴别中的应用价值。方法以上海市肺科医院1998年1月至2003年12月住院患者76例为研究对象，其中结核病组30例、结节病组31例和正常对照组（其他疾病）15例，利用定量PCR检测用石蜡包埋的淋巴结或肺组织中结核分枝杆菌DNA，并以胎鼠肺组织作为阴性对照。结果结核病组结核分枝杆菌DNA检出的阳性率（30／30）明显高于结节病组（6／31）和正常对照组（2／15），结节病组与正常对照组结核分枝杆菌DNA检出的阳性率无明显差别。结核病组结核分枝杆菌DNA检出的绝对和相对拷贝数明显高于结节病组和正常对照组，结节病组与正常对照组无明显差别，而胎鼠肺组织中未检出结核分枝杆菌DNA。结论本研究结果不支持结核分枝杆菌感染与结节病的相关性。实时定量PCR法检测石蜡包埋病理组织中结核分枝杆菌DNA可作为鉴别增殖性结核和结节病的方法之一。