聚合酶链反应在诊断结核性胸腔积液中的应用

应用聚合酶链反应（ＰＣＲ）技术对６２例胸水进行结核菌ＤＮＡ检则，并同时与胸水涂片及培养结果进行比较。结果显示：３４例结核性胸水涂片抗酸染色、结核菌培养和ｐＣＲ检测阳性率分别为５．９％、８．８％和５２．９％，后者显著高于前二者（Ｐ均＜０．０１）。２８例非结核性胸水涂片和培养结果均呈阴性，但ＰＣＲ有４例（１４．３％）出现阳性。结果表明ＰＣＲ直接检测胸水中结核菌显示出快速、敏感和高效等优点。同时还对影响ＰＣＲ检测结核菌的某些因素作了分析。     

聚合酶链反应测Vi抗原在伤寒早期诊断中的应用

为选择更为敏感、特异的伤寒早期诊断方法，从伤塞沙门菌表达Ｖｉ抗原的特异结构基因ＶｉａＢ区域选择二对引物，建立了套式－聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）。检测伤寒早期标本３６份、结果血培养阳性２５份（６９％），肥达反应阳性１７份（４７．２％），ＰＣＲ阳性２９份（８０．６％）；２５份血培养阳性标本，ＰＣＲ均为阳性，并从血培养奶性标本中检出Ｖｉ－ＤＮＡ阳性４份。研究表明，应用ｎｅｓｔ－ｅｄ－ＰＣＲ检测     

巢式聚合酶链反应

巢式聚合酶链反应山西医学院王守义医师函问：在聚合酶链反应（ＰＣＲ）技术中什么叫巢式？什么叫外引物、内引物？侯金林、骆抗先医师（第一军医大学南方医院，广州５１０５１５）答：巢式ＰＣＲ（Ｎｅｓｔｅｄ－ｐｒｉｍｅｒｓＰＣＲ）是通过“外”、“内”两对引物完成...     

TTV在非甲-庚型肝炎患者中的检测、克隆及测序

目的分析ＴＴＶ（Ｔｒａｎｓｆｕｓｉｏｎｔｒａｎｓｍｉｔｅｄｖｉｒｕｓ）深圳分离株ＯＲＦ１部分基因序列。方法在ＴＴＶＯＲＦ１设计引物，建立巢式聚合酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ），检测４０例广东地区非甲－庚型肝炎患者血清中ＴＴＶＤＮＡ。对ＰＣＲ产物进行分子克隆，以荧光法（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ，３７３Ａ）测序。结果４０例非甲－庚型肝炎中２１例ＴＴＶＤＮＡ阳性（５２．５％）。对其中一株ＴＴＶ（ＳＺ１）ＯＲＦ１部分基因克隆、测序，并与Ｏｋａｍｏｔｏ等报道的２株（Ｎ２２、Ｇ１ａ）相比较，其核苷酸序列的同源性分别为９６．７％与９７．４％。结论本研究证实我国华南地区存在ＴＴＶ感染。ＴＴＶ的分子克隆与测序及ＰＣＲ诊断技术的建立对国内进一步开展ＴＴＶ感染的诊断与流行病学调查均具有重要意义     

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。     

逆转录—套式—聚合酶链反应检测HCV RNA正链和负链及与肝细胞…

为了解ＨＣＶ与肝癌的关系，采用ＨＣＶ５′－ＮＣＲ正相引物和反相引物逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测了２２例肝癌手术患者血清，癌组织和癌旁组织ＨＣＶＲＮＡ正链和负链，用ＨＢＶＣ保守区引物ＰＣＲ法检测了ＨＢＶＤＮＡ，结果发现，５例抗－ＨＣＶ阳性的肝癌患者中，３例血清，癌组织和癌旁组织ＨＣＶＲＮＡ正链扩增试验均阳性，１例三者均阴性，１例血清和癌旁组织阳性。３例正链扩增试验阳性     

基因型和核苷酸序列分析研究丙型肝炎病毒母婴传播

为研究丙型肝炎病毒（ＨＣＶ）的母婴传播，应用ＥＩＡ方法检测１８１６例孕妇血清抗－ＨＣＶ抗体。结果抗体阳性者１６例（０．８８％），进一步用逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测病毒核酸（ＨＣＶＲＮＡ）１４例孕妇为阳性，对其中１３例孕妇所生１３例婴儿随访至出生后６个月，３例婴儿检出ＨＣＶＲＮＡ，其中２例为短暂ＨＣＶ感染者，１例为慢性ＨＣＶ感染者伴ＡＬＴ异常，母婴传播率为２３．０％     

聚合酶链反应诊断结核性腹膜炎的评价

目的评价聚合酶链反应（ＰＣＲ）方法对结核性腹膜炎的诊断价值。方法用ＰＣＲ技术检测３０例结核腹水中结核分支杆菌ＤＮＡ，并与腹水涂片抗酸染色及酶联免疫吸附试验（ＥＬＩＳＡ）检测腹水中抗ＰＰＤ抗体进行比较。结果ＰＣＲ的阳性率为６０％，特异性９４．４％；ＥＬＩＳＡ法阳性率６３．３％，特异性７２．２％；涂片镜检均为阴性。结论ＰＣＲ在诊断结核性腹膜炎具有较高的敏感性和特异性，优于ＥＬＩＳＡ法及涂片镜检，如与ＥＬＩＳＡ技术结合可进一步提高检测的敏感性和特异性。     

聚合酶链反应试剂对检测痰结核分支杆菌的影响

目的观察聚合酶链反应（ＰＣＲ）的试剂对检测痰结核分支杆菌的敏感性和特异性的影响。方法对结核病组２０５例和非结核病组１０５例的痰标本同时进行三个厂家生产的ＰＣＲ试剂检测、普通细菌培养和抗酸染色法涂片检测。再对ＰＣＲ假阳性的痰普通细菌培养结果进行分析，试找出ＰＣＲ假阳性的其它因素。结果三种不同引物序列和扩增产物片段长度的ＰＣＲ试剂对结核性痰标本的阳性率分别为８２．４％、７１．７％和６１．０％，组间差异有非常显著意义（Ｐ＜０．００５）。结论ＰＣＲ试剂的引物序列和扩增产物长度对检测痰结核分支杆菌的阳性率和假阳性率有很大的影响     

聚合酶链反应在诊断结核性胸膜炎中的应用

目的：应用ＰＣＲ技术对７０例胸水进行结核菌ＤＮＡ检测。方法：７０例胸水做ＴＢ－ＰＣＲ、胸水抗酸菌涂片，皮肤结素试验及血结核菌抗体检测，并与临床诊断标准进行比较。结果：７０例胸水中，结核性胸腔积液４６例，ＰＣＲ阳性率４５．７％，涂片镜检、结素强阳性、结核菌抗体（＋）则分别为２．２％，８．７％，１０．９％，前者明显高于后三者（Ｐ值均＜０．００５），但其ＰＣＲ敏感性与临床诊断标准比较相关性仍低，非结核性胸水２４例中，ＰＣＲ阳性率８．３％。结论：ＰＣＲ检测结核菌尚有一些条件需进一步完善，目前临床尚不能将其作为诊断结核病包括结核性胸膜炎的标准，仍需结合病人的临床表现及其它检查结果来综合判断。     

结核性腹膜炎的实验室诊断

目的评价聚合酶链反应（ＰＣＲ）结合Ｓｏｕｔｈｅｒｎ杂交技术及酶联免疫吸附试验（ＥＬＩＳＡ）对结核性腹膜炎的诊断价值。方法用ＰＣＲ结合地高辛标记核酸探针Ｓｏｕｔｈｅｒｎ杂交技术检测４２例结核性腹水中结核分支杆菌ＤＮＡ，并与常规细菌学检测及ＥＬＩＳＡ对比。引物来自结核分支杆菌特异重复插入序列ＩＳ６１１０。特异性通过杂交及限制性内切酶ＳａｌⅠ酶切证实。同时比较了Ｓｏｕｔｈｅｒｎ杂交检测与凝胶电泳检测的敏感性。结果ＰＣＲ的敏感性为６９％，ＥＬＩＳＡ为７１％，培养为９％，涂片镜检均为阴性。并发现杂交较凝胶电泳检测更敏感。结论ＰＣＲ和ＥＬＩＳＡ法对结核性腹膜炎有较高的诊断价值，但前者更具有特异性。将Ｓｏｕｔｈｅｒｎ杂交技术与ＰＣＲ技术结合，可进一步提高检测的敏感性和特异性。     

Polymerase chain reaction of pleural biopsy is a rapid and sensitive method for the diagnosis of tuberculous pleural effusion

BACKGROUND: Tuberculous pleural effusion occurs in 30% of patients with tuberculosis (TB). Rapid diagnosis of a tuberculous pleural effusion would greatly facilitate the management of many patients. Polymerase chain reaction (PCR) has been used to detect Mycobacterium tuberculosis in pleural fluid with highly variable sensitivity. OBJECTIVE: To improve our laboratory diagnosis of tuberculous pleural effusion. METHODS: We applied PCR to detect DNA specific for M tuberculosis in 33 of the studied pleural biopsy specimens using an IS986-based primer that was specific for mycobacterium complex, and compared it to the results of pleural fluid and biopsy cultures performed on either Lowenstein-Jensen (LJ) medium or BACTEC 12B liquid medium (Becton Dickinson Microbiology Systems; Cockeysville, MD), Ziehl-Neelsen (ZN) staining, and histopathology in 45 patients with pleural effusion. RESULTS: Of the 45 patients with pleural effusion who were studied, 26 patients received diagnoses of tuberculous pleural effusion that had been confirmed by either culture and or histopathology, 10 patients received diagnoses of exudative effusion due to causes other than TB, and 9 patients received diagnoses of transudative effusion. Histopathology of the pleural biopsy specimen had a sensitivity of 53.8%. The sensitivity of the ZN staining of pleural fluid and biopsy specimens was 0.0% and 3.8%, respectively. The sensitivity of the culture on both BACTEC 12B liquid medium and LJ medium was higher in pleural biopsy specimens (92.3%) than in pleural fluid specimens (15.4%; p > 0.001). The improvements of the BACTEC culture system improved and shortened the detection time of M tuberculosis in pleural biopsy specimens. PCR of pleural biopsy specimens had 90% sensitivity and 100% specificity. The positive predictive value and the negative predictive value for pleural biopsy specimen cultures were 100% and 90.5% vs 100% and 86.7% for pleural biopsy specimen PCRs. CONCLUSION: The overall accuracy of PCR of pleural biopsy was similar to the results of pleural biopsy culture, however, PCR of the pleural biopsy was much faster in reaching diagnosis. PCR of pleural biopsy is a useful method when used in combination with the BACTEC culture system and histopathologic examination of pleural biopsy to reach a rapid diagnosis of tuberculous pleural effusion.     

Rapid diagnosis of tuberculous pleural effusion using polymerase chain reaction

Between October 1998 and September 1999, 98 patients with symptomatic exudative lymphocytic pleural effusion were enrolled in our study to evaluate the diagnostic sensitivity of polymerase chain reaction (PCR) assay. The mean age was 53.3 years ranging from 18 to 78 years. There were 61 men and 37 women. Pleural fluid was sent for gram staining, AFB staining, aerobic culture, culture of Mycobacterium tuberculosis on LJ media, and cytology. Additional fluid was used for a PCR-assay of the 16 S-23 S rRNA gene spacer sequences and for a nested PCR of the 16 S rRNA gene as a blind control. In cases of free-flow pleural tapping, histopathological analysis was done on three pleural biopsies. Overall etiologies comprised malignancy 53.1%, tuberculosis 36.7%, lymphoma 2.0% and chronic nonspecific inflammation 8.2%. The sensitivity and specificity of AFB-staining were 6% and 79%, respectively; while cultures on LJ media were 17% and 100%, respectively. The sensitivity of the PCR-assay was 50% (95% CI: 40 to 60%) and the specificity was 61% (95 CI: 52 to 71%). When PCR was nested, the sensitivity was 72% (95% CI: 63 to 81%) and specificity was 53% (95% CI: 43 to 63%). Two thirds (26 of 36) of tuberculous pleural effusion cases underwent pleural biopsy, and 62% were diagnosed by histopathology. There were no complications from thoracocentesis or pleural biopsy in any of the patients. We concluded that PCR assay was more sensitive than AFB staining and mycobacteria culture for diagnosis tuberculous pleural effusion but its specificity was quite low.     

不同检测方法在结核性脑膜炎诊断中的应用价值

搜集陕西省结核病防治院2015年5月至2017年5月疑似结核性脑膜炎的住院患者519例,最终根据2010年国际结核性脑膜炎协作组制定的诊断标准,354例被诊断为结核性脑膜炎,其余165例排除结核性胸膜炎的诊断。评价BACTEC MGIT 960液体快速培养(简称“MGIT 960培养”)、RNA恒温扩增实时荧光检测技术(SAT-TB技术)、聚合酶链式反应-荧光探针检测技术(简称“PCR技术”)、GeneXpert MTB/RIF(简称“GeneXpert”)技术对结核性脑膜炎的诊断价值。MGIT 960培养、SAT-TB技术、PCR技术、GeneXpert技术检测结核性脑膜炎的敏感度及其95%CI值分别为23.7%(84/354;19.5%~28.4%)、7.6%(27/354;5.2%~10.7%)、12.4%(44/354;9.3%~16.2%)、39.5%(140/354;34.6%~44.7%),特异度分别为100.0%(165/165)、100.0%(165/165)、100.0%(165/165)、99.4%(164/165)。GeneXpert技术检测的敏感度明显高于MGIT 960培养、SAT-TB和PCR技术。     

Diagnosis of tuberculosis in children using a polymerase chain reaction.

We investigated the value of the polymerase chain reaction (PCR) in the diagnosis of active tuberculosis in children and evaluated the relationship between PCR results in children with tuberculous infections and mediastinal adenopathies detected by computerized tomography (CT-Scan). This was a controlled, blinded, prospective study comparing nested PCR, mycobacterial cultures and the clinical diagnosis based on 350 clinical specimens from 117 children referred for evaluation of suspected pulmonary tuberculosis. All children with tuberculous infection but without active disease underwent a chest CT-scan to detect the presence of mediastinal adenopathies not evident on chest x-ray. The sensitivity of PCR was 56.8% in children with clinically active disease (culture: 37.8%; smears: 13.5%). A major advantage of PCR over cultures was noted when there was no parenchymal involvement on chest radiograph and when the patient was undergoing anti-tuberculous treatment. There were nine specimens with false-negative PCR results due to the presence of amplification reaction inhibitors. PCR was positive in five children with tuberculous infection without active disease and these children presented mediastinal adenopathies on the CT-scan that were not evident on chest radiography. There were no false-positive PCR results in the control groups of children. We conclude that nested PCR is a rapid and sensitive method for the early diagnosis of tuberculosis in children. It is especially useful when the diagnosis of active tuberculosis is difficult. In our study children with tuberculous infection without apparent disease who have positive PCR results have mediastinal adenopathies on CT-scan.     

Searching for somatic mutations in McCune-Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples

BACKGROUND: Activating mutations of the Gsalpha gene (GNAS), which encodes for the alpha-subunit of the stimulatory G protein, have been identified in patients with McCune-Albright syndrome (MAS). Accuracy and sensitivity in the molecular diagnosis of MAS is mandatory for optimal therapeutic strategy and adapted follow-up, especially for incomplete clinical forms of MAS. To date, the highly sensitive nested PCR method with intermediary digestion by a restriction enzyme at the mutation site is one of the most widely used techniques. This study evaluated a new diagnostic method using a peptidic nucleic acid (PNA) and compared it with the nested PCR method. Material and methods: One hundred and forty-eight DNA samples from eighty-eight patients presenting clinical symptoms compatible with MAS were included. The DNA samples were mainly obtained from peripheral blood, ovarian tissue or cyst liquid, and bone lesions. The nested PCR method required 4 days. PNA clamping required 1.5 days and utilized the higher thermal stability and specificity of PNA-DNA coupling to inhibit PCR product formation. Direct sequencing was subsequently performed in all cases. RESULTS: The sensitivity of mutation detection was 54% (n = 80) for nested PCR and 46.6% (n = 69) for PNA (P > 0.05). The 11 cases where PNA failed to detect the mutation were mainly incomplete and atypical clinical forms of MAS (n = 10/11). The cost per sample was 50 Euros for PNA clamping versus 136 Euros for nested PCR. CONCLUSION: PNA clamping is a rapid, reliable, and economical method to diagnose MAS. It should be the first-line diagnostic method, although negative results, especially for incomplete clinical forms of MAS, should be confirmed by nested PCR.     

免疫组织化学及PCR技术在淋巴结结核病理诊断中的应用价值

目的 探讨免疫组织化学(immunohistochemistry,IHC)及PCR技术在淋巴结结核病理学诊断中的应用价值。方法 收集首都医科大学附属北京胸科医院病理科保存的2012年1月至2013年7月之间的48例手术根治切除淋巴结结核患者(结核组)和21例非淋巴结结核患者(非结核组)的石蜡包埋组织标本,分别应用IHC染色、荧光定量PCR和抗酸染色法对标本进行检测,以临床最后诊断为金标准,比较各方法的检测效能。结果 IHC染色、荧光定量PCR及抗酸染色检测的敏感度分别为52.1%(25/48)、60.4%(29/48)及27.1%(13/48);IHC染色和荧光定量PCR检测敏感度均高于抗酸染色,差异均有统计学意义(χ ²值分别为 6.27、10.84,P值分别为0.012、0.001);而IHC染色与荧光定量PCR比较,敏感度差异无统计学意义(χ ²=0.68,P=0.411)。IHC染色、荧光定量PCR及抗酸染色法检测非结核组结果均为阴性,特异度均为100%(21/21)。IHC染色、荧光定量PCR及抗酸染色法的阴性预测值分别为47.7%(21/44)、52.5%(21/40)、37.5%(21/56);符合率分别为66.7%(46/69)、72.5%(50/69)、49.3%(34/69),IHC染色、荧光定量PCR均优于抗酸染色法。 结论 IHC染色、荧光定量PCR与抗酸染色法相比,可提高阳性检出率,在淋巴结结核的病理学诊断中具有良好应用价值。     

Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.