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     

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。     

聚合酶链反应检测福尔马林固定、石蜡包埋肝穿刺活检组织中HBV DNA

本文应用聚合酶链反应(PCR)技术检测了21例慢性肝炎患者福尔马林固定、石蜡包埋肝穿活检组织中的HBV DNA,并与其中的16例新鲜肝组织HBV DNA检出率进行了对照研究。结果显示:福尔马林固定、石蜡包埋肝穿刺活检组织HBV DNA检出率为71.43％(15/21),新鲜肝穿组织中HBV DNA检出率为87.50％(14/16),两者比较P>0.05。本文结果表明:(PCR)技术检测福尔马林固定、石蜡包埋肝穿刺活捡组织中的HBV DNA,具有材料来源广,易于保存等优点,适用于回顾性研究。     

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     

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 …

探讨外周血单个核细胞中结核分支杆菌ＤＮＡ检测在肺结核诊断中的价值。方法采用改良Ｔｒｉｔｏｎ－ｘ－１００法分离，制备单个核细胞中模板，ＤＮＡ，聚合酶链反应扩增结核分支杆菌２４０ｂｐ基因片段，同时分析了影响ＰＣＲ结果的有关因素。结果８９例肺结核患者的血标本，８４例肺结核患者的痰标本中，结核分支杆菌ＤＮＡ阳性率分别为７３％和５７％；８４例肺结核患者外周血，痰标本配对检测总阳性率可达８７％。     

DNA扩增抑制试验在聚合酶链反应检测结核分支杆菌中的作用

ＤＮＡ扩增抑制试验在聚合酶链反应检测结核分支杆菌中的作用张显忠郭爱军曹洪林杨明峰我们建立了ＤＮＡ扩增抑制试验，检测系统中抑制因素所造成聚合酶链反应（ＰＣＲ）试验的假阴性，以使ＰＣＲ技术更加完善。材料与方法：标本来源：病例系本院住院肺结核病患者，根据临...     

聚合酶链反应检测乙型肝炎患者心肌组织中HBV DNA

应用巢式聚合酶链反应（ＰＣＲ）技术检测了１８例乙型肝炎（乙肝）患者石蜡包埋心、肝组织中的ＨＢＶ ＤＮＡ，并与其免疫组化及原位杂交结果作了比较。二组引物检测的结果表明：在肝组织中均有ＨＢＶ感染，其中５例有ＨＢＶ复制。心肌组织中ＨＢＶ ＤＮＡ的检出率为５５．６％，不存在ＨＢＶ的复制。心脏病理检查可见一些非特异性改变，如脂变、水肿和纤维素样坏死等。病理变化与心电图、临床症状、体征及ＰＣＲ检测结果无相关性     

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.     

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。     

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.     

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。     

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.     

利用聚合酶链反应检测122例结核病患者标本中结核菌的研究

对１２２例门诊及住院结核病人的痰、脓汁等标本进行聚合酶链反应（ＰＣＲ）和直接涂片荧光镜检，培养检查结核菌，结果表示：ＰＣＲ阳性率为５６．９％（５８／１０２），培养阳性率为１４．７％（１５／１０２），直接涂片荧光镜检为２３．５％（２４／１０２）。说明ＰＣＲ特异性好，敏感性强，与临床符合率较高。ＰＣＲ比培养法大大节省时间，为结核病的诊断和鉴别赢得了时间，是一种可以推广的好方法。     

16SrRNA基因PCR加反相杂交技术检测细菌DNA

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本细菌ＤＮＡ。结果对２０株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人基因组及病毒无交叉反应；２２份血培养阳性标本及４份脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据。