聚合酶链反应检测结核患者各种标本的比较

用聚合酶链反应检测１２０例结核患者各种标本阳性率为４３．３％，而非结核患者为０，说明ＰＣＲ方法敏感性好，特异性好，经检测各种标本及比较，发现以痰、脓汁，胸腹水检出阳性率高，而痰取材方便，病人乐于接受，便于临床推广。     

P53基因PCR-SSCP银染检查痰标本诊断肺癌初步探索

目的：为了解痰标本检测肺癌患者Ｐ５３基因突变的可行性及其对临床诊断的意义。材料与方法：采用改良Ｓａｃｃｏｍａｎｎｏ法浓缩及保存痰标本,部分涂片找癌细胞,其余标本进行痰细胞ＤＮＡ提取及多聚酶链反应（ＰＣＲ）－单链构象多态性分析（ＳＳＣＰ）共检测２２例病人,其中确诊肺癌病人１５例（涂片或活检找到癌细胞）、临床高度怀疑肺癌病人５例（涂片或活检均阴性）、２例非肿瘤病人（１例为结节病,另１为左中叶肺炎）,同     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

套式PCR检测婴幼儿巨细胞病毒感染

本文采用套式ＰＣＲ（Ｎｅｓｔｅｄｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）技术对６１例婴儿肝炎综合征（Ｎｅｏｎａｔａｌｈｅｐａｔｉｔｉｓ）患者进行了ＨＣＭＶ（Ｈｕｍａｎｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ）检测,结果表明,当只用一对引物进行ＰＣＲ时,检出３３例患者为ＨＣＭＶ阳性,阳性检出率为５４．１％;而采用套式ＰＣＲ后,有１７例为ＨＣＭＶ阳性,阳性检出率提高到７７．０％。可见套式ＰＣＲ方法的阳性检出率明显高于单一ＰＣＲ,并远高于目前使用的其它方法。同时也说明ＨＣＭＶ是导致肝炎综合征的主要病原体之一。此外,灵敏性和特异性试验表明,套式ＰＣＲ技术的敏感度高、特异性强、简单快速,在临床检测和诊断中具有很大的潜力。     

PCR技术在三种男性性病检测中的应用

本文用聚合酶链反应ＰＣＲ技术对３６９例男性泌尿生殖道炎患者进行淋球菌（ＮＧ）,解脲支原体（ＭＰＵ）和沙眼衣原体（ＣＴ）的检测,阳性率分别为３５．５％,３０．５％和１１．６％,同时,我们对高度怀疑为淋病患者７４例及６３例非淋病性尿道炎患者进行细菌培养和ＰＣＲ的同时检测,淋病患者细菌培养阳性检测率为３１．１％,与９２-９４年间所进行的培养检测结果基本一致,均低于ＰＣＲ检测结果（７７．１％）,解脲支原体培养阳性率为１７．５％,亦明显低于ＰＣＲ结果（３０．５％）,此外,对同一患者进行ＮＧ＋ＭＰＵ和ＭＰＵ＋ＣＴＰＣＲ检测,发现约４０％的淋病患者并发ＭＰＵ感染,而ＭＰＵ和ＣＴ复合感染亦在１０％左右。上述结果说明ＰＣＲ方法在性病病原体的诊断中比细菌培养法更为可靠,特别是在多种病原体复合感染情况下更为有效。     

应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

应用PCR-SSCP快速鉴定结核分枝杆菌复合群

通过聚合酶链反应（ＰＣＲ）－单链构象多态性（ＳＳＣＰ）技术分析结核分枝杆菌和非结核分枝杆菌临床株的１６ＳｒＤＮＡ基因。６０例临床标本中,２０例为阳性,与传统方法比较无差异。其中分型：１８例为结核分枝杆菌,２例为结核分枝杆菌和非结核分枝杆菌双重感染。２０例阴性标本中,ＰＣＲ－ＳＳＣＰ又检查出５例阳性。２０例对照标本中,３种传统方法与ＰＣＲ－ＳＳＣＰ法均检测为阴性。全部试验３ｄ报告结果。结核分枝杆菌复合群和卡介苗的图形相同以外,其它分枝杆菌的ＰＣＲ－ＳＳＣＰ电泳图谱均有差异。所以,应用ＰＣＲ－ＳＳＣＰ技术快速     

用PCR快速检测胃活检标本中的幽门螺杆菌

本文用ＰＣＲ对１１６份胃活检标本进行幽门螺杜菌（ＨｅｔｉｃｏｂａｃｔｅｒＰｙｌｏｒｉＨｐ）检测,总阳性率７１．５５％（８３／１１６）,胃溃疡、十二指肠溃疡和胃炎的阳性率分别为８７．５％（１４／１６）、８６．９６％（２０／２３）和６３．５１％（４７／７４）,同时用尿素酶法作参照,其总阳性率为５８．６２％（６８／１１６）,上述疾病分别为４３．７５％（７／１６）、５６．５２％（１３／２３）和６２．１６（４６／７４）。结果表明ＰＣＲ能快速、敏感特异地检出ＨＰ,这对研究ＨＰ在胃、十二指肠疾病中的致病作用、传播途径及疗效观察具有重要意义。     

超高忠实性PCR用DNA聚合酶

ＰＣＲ（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ）法已在生物工程领域得到了广泛的应用,而ＰＣＲ法的推广完全得益于Ｔａｑ耐热性ＤＮＡ聚合酶。近年来,ＰＣＲ法的应用除生物工程领域外,已应用至工业、农业、医学、制药等与人们生活息息相关的各个领域。随着ＰＣＲ法的不断推广,人们对ＤＮＡ聚合酶的忠实性（Ｆｉｄｅｌｉｔｙ）要求越来越高,一般来说,ＴａｑＤＮＡ聚合酶在进行ＰＣＲ延伸反应过程中的错配率为０．５％左右。ＴａＫａＲａ公司独自研制成功的ＰｙｒｏｂｅｓｔＤＮＡ聚合酶是一种来源于Ｐｙｒｏｃｏｃｃｕｓｓ…     

应用PCR—SSCP快速鉴定结构分枝杆菌复合群

通过聚合酶链反应（ＰＣＲ）－单链的权象多态性（ＳＳＣＰ）技术分析结构分枝杆菌和非结构分枝杆菌临床株的１６Ｓ ｒＤＮＡ基因。６０例临床标本中,２０例为阳性,与传统方法比较无差异。其中分型：１８例为结核支杆菌,２例为结核分枝杆菌和非结核分枝杆菌双重感染。２０例阴性标本中,ＰＣＲ－ＳＳＣＰ又检查出５例阳性。２０例对照标本中,３种传统方法与ＰＣＲ－ＳＳＣＰ法均检测为阴性。全部试验３ｄ执行结果。结核分枝杆菌     

Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

运用液相芯片技术原理,以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因,设计筛选4套扩增引物和杂交探针,建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示,四重液相芯片方法可特异检测鉴别目标菌种(群),与其它分枝杆菌菌种或微生物无非特异交叉反应;检测敏感性达2.1×101-2.5×102基因拷贝或0.06-0.74 fg DNA;组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌,检出率分别达75.6%(99/131)和94.9%(37/39),显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示,四重液相芯片方法与荧光PCR方法的阳性符合率为83%(24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d,其中对纯化DNA模板的检测时间可在2-3 h内完成。     

应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感…     

Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7

Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.     

In vitro efficacy of Linezolid on clinical strains of Mycobacterium tuberculosis and other mycobacteria

Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.     

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.     

Cytochrome CYP141: a new target for direct detection of Mycobacterium tuberculosis from clinical specimens

Cytochrome P450 CYP141 is an intermediary metabolic and respiratory protein that interferes with oxidation reduction in Mycobacterium tuberculosis. This conserved protein has also been debated as a hypothetical target for therapeutics. We used the sequences of CYP141 gene to develop a PCR for rapid detection of Mycobacterium tuberculosis from respiratory specimens. The sensitivity of this PCR for culture positive-smear positive and culture positive-smear negative samples were 92% and 62.5%, respectively. The overall sensitivity and specificity of this PCR was 85.7% and 97.8%. As compared with other studies, it appears that the CYP141 gene is a good target for direct detection of M. tuberculosis from respiratory specimens.     

Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis

A simple latex agglutination test (SLAT) based on modifications of existing serodiagnostic techniques, in which commercially available reagents are used, was developed for detection of antibodies against Mycobacterium tuberculosis. Tests performed on 553 serum samples from 316 individuals, including 117 bacteriologically confirmed active tuberculosis patients, showed 80% positive titers. Sera from 12 patients with arrested tuberculosis showed 91% positive titers. Nonspecific reactions were noted in 5% of 160 serums from selected normal individuals and patients with diseases other than tuberculosis. The antibodies detected by the SLAT method were found to be relatively stable when exposed to low temperatures, whereas high temperatures reduced the antibody titer considerably. Disodium ethylenediaminetetraacetic acid inactivation of serum complement was found to be satisfactory. No variation of tuberculosis antibody titer was noted in tests on multiple specimens from patients whose conditions were stabilized. However, considerable fluctuation was encountered in antibody titers obtained on recently detected individuals. Data obtained in this study indicate that the modified procedures of the SLAT method could replace the tuberculin skin test for simple screening of tuberculosis in adults.     

结核分枝杆菌重组38kD蛋白用于新疆南疆维吾尔族和 湖南湘中汉族血清学诊断的研究

目的：对结核分枝杆菌38kD 蛋白编码基因进行克隆表达及纯化,建立基于重组38 kD 蛋白的酶联免疫吸附法(ELISA)检测 结核病人血清标本,评价重组38 kD蛋白用于结核病血清学诊断抗原的价值。并比较分析其在汉族和维吾尔族人群中的血清学 诊断的差异。方法：用PCR方法扩增38 kD 蛋白的编码基因, 构建重组质粒, 转化到大肠杆菌BL21 中,经IPTG诱导表达, 得到纯 化的38 kD 蛋白,建立以38 kD蛋白为包被抗原的ELISA,并检测临床确诊的结核病人血清标本。结果：ELISA 检测结核病患者 血清标本的维吾尔族阳性率为34%（52/153）,汉族为52.4%（65/124）,两者对比有统计学差异（X2＝9.538,P＜0.005）。在阴性对照 中的维吾尔族特异度为96.4%（159/165）,汉族为98. 8%（130/133）,结果无统计学意义（X2＝0.111,P＞0.5）。结论：重组38kD 蛋白 用于血清学诊断的敏感度在维吾尔族和汉族中有差异,而其诊断特异度无差别。