PCR技术在检测活检组织中结核杆菌的应用

ＰＣＲ技术在检测活检组织中结核杆菌的应用潘美华作者单位：安徽医科大学附属医院病理科，合肥２３００２２在临床诊断结核病中，用ＰＣＲ技术检测痰液、血液、脑脊液、胸腹水等样品中的结核杆菌已得到了广泛的应用，但在病理诊断中所遇到的一些组织内结核病变不典型，而...     

PCR技术检测活检组织中结核杆菌DNA

ＰＣＲ技术检测活检组织中结核杆菌ＤＮＡ赵跃武银平章刘正国郝远瑞徐纪跃党伟一作者单位：河南省人民医院病理科，郑州４５０００３多聚酶链反应（ＰＣＲ）是一项有效的体外ＤＮＡ扩增技术，尤在病原体检测方面。在一些可疑的活检石蜡切片中，有时也需要借助于该技术来协...     

多聚酶链反应（PCR）检测不典型结核杆菌的DNA

本文采用ＰＣＲ技术检测结核杆菌的ＤＮＡ（ＴＢ－ＤＮＡ），其中临床诊断为结核或可疑结核者９例，肿瘤６例，结节病３例，淋巴结炎４例，共２２例。病理诊断确诊为不典型结核者１４例，不能除外结核者４例，结节病２例，瘤样病变２例。所有病例经抗酸染色均未查到结核杆菌。ＰＣＲ检测结果表明，２２例中１８例呈阳性（占８１．８２％），４例呈阴性。ＰＣＲ技术敏感、特异，对于不典型结核病例检查ＴＢ－ＤＮＡ片段用于辅助诊断颇     

聚合酶链反应技术快速检测肺组织中结核杆菌的研究

聚合酶链反应技术快速检测肺组织中结核杆菌的研究庄玉辉，李宁，张小刚，李国利，吴雪琼，张玉芝有些结核病例在病理组织学上往往缺乏典型的结核肉芽肿和干酪样坏死，与结节病肉芽肿、肺部圆形病灶等组织切片不易鉴别，常常依赖于病原学检查。传统的病原学诊断方法敏感性...     

PCR检测结核杆菌DNA的临床应用

聚合酶链反应(PCR)自1989年引入结核病诊断以来,已成为结核病细菌学诊断中关注的焦点.为结核病的早期诊断、治疗、预防开拓了新领域,因此我们采用聚合酶链反应技术检测结核菌特异性DNA片段,现报告如下.     

原位PCR技术

原位ＰＣＲ是一种通过在单细胞或组织切片上对特异的核酸序列进行原位扩增，检测定位的新技术，本文对其基本的原理、发展简史、基本步骤及应用当中的注意事项进行了综述。     

用巢式PCR 对孕妇外周血中的单个有核红细胞进行胎儿性别诊断

目的 建立利用孕妇外周血中的单个有核红细胞诊断胎儿性别的方法。方法 46名中期妊娠妇女外周血经密度梯度离心后瑞氏染色，显微操作分离单个有核红细胞，并用巢式PCR扩增SRY基因。结果 胎儿性别的总符合率为82.61%，敏感性为80％，特异性为87.50%。结论 利用孕妇外周血中的单个有核红细胞诊断胎儿性别的方法具有一定的可行性。     

毛细管PCR技术在传染病快速诊断上的初步应用

常规ＰＣＲ反应需要２～６小时，而在毛细管ＰＣＲ反应中，由于采用毛细管作为反应容器，因此能在１５分钟完成３０个循环。对国产毛细管ＰＣＲ仪进行优化的结果表明：循环参数为９４℃０秒（即＜１秒），５０℃～５５℃０秒，７２℃２０秒，ＰＣＲ反应达到最佳。敏感性实验表明：常规ＰＣＲ可检测１．０ｆｇＨＢＶＤＮＡ，毛细管ＰＣＲ可检测０．１ｆｇＨＢＶＤＮＡ。用１１种引物从多种标本中成功地扩增了９种病原微生物相应基因片段。建立了ＫＩ－玻璃粉法快速制备ＰＣＲ模板的方法，几乎适合所有类型的临床标本。将毛细管ＰＣＲ用于检测血清中ＨＢＶＤＮＡ，结合快速热处理法制备ＰＣＲ模板，可在１．５小时内对２０例标本报告结果。毛细管ＰＣＲ技术的建立，为传染病的基因诊断向快速化发展开辟了新的途径。     

用PCR制备地高辛精标记的单链探针进行原位杂交

用聚合酶链反应（ＰＣＲ）方法制备地高精标记的单链探针，用以原位检测低丰度的β肾上腺素能受体ＭＲＮＡ在大鼠肺组织中扮布。结果表明本方法制备的探针的检测敏感性显著高于用随机引物法标记的双链探针。已克隆于载体的ＣＤＮＡ或ＲＴ－ＰＣＲ产物均可作为模板合成单链探针，江ＤＡＴＰ、ＤＣＴＰ、ＤＧＴＰ浓度各为２００μｍｏｌ／Ｌ，ｄＴＴＰ＋Ｄｉｇ－ｄＵＴＰ浓度降至５０－７５ μｍｏｌ／Ｌ，扩增产量无显著变化，ｄＴＴ     

PCR检查女性无症状淋病

用ＰＣＲ法和涂片染色法检查３０例无症状淋病患者和５０例有症状淋病患者，结果：查有症状淋病，ＰＣＲ法和染色涂片法无显著性差异，查无症状淋病，ＰＣＲ法和染色涂片法有显著性差异。认为，淋球菌感染初期，治疗后的复查及症状不典型患者，ＰＣＲ法能较好地防止漏检。     

组织学三联征和聚合酶链反应在弓形虫淋巴结炎病理诊断中的应用

目的 经典的组织学三联征诊断法和荧光原位杂交(PCR)法在提高石蜡包埋组织弓形虫淋巴结炎确诊率中的价值.方法 收集本院1999年4月至2009年9月诊断的46例符合组织学三联征形态改变的石蜡包埋淋巴结组织,采用半嵌套式PCR方法 对提取DNA进行弓形虫基因组的片段扩增;另选取30例组织中可见三联征中的二个或一个特征的病例作为对照.结果 组织学三联征组PCR阳性率为76.1%(35/46),对照组阳性率仅为10.0%(3/30,P<0.01);组织学三联征诊断弓形虫淋巴结炎的灵敏度为92.1%(35/38),特异度为71.1%(27/38);PCR法的阳性预测值为76.1%(35/46),阴性预测值为90.0%(27/30).结论 经典的组织学三联征对于诊断弓形虫淋巴结炎的特异性很强,但敏感性较低,且易漏诊部分非典型病例.在组织形态改变的基础上结合半嵌套式PCR对刚地弓形虫基因片段的检测可大大提高检出敏感性和准确性.     

组织学三联征和聚合酶链反应在弓形虫淋巴结炎病理诊断中的应用

目的 经典的组织学三联征诊断法和荧光原位杂交(PCR)法在提高石蜡包埋组织弓形虫淋巴结炎确诊率中的价值.方法 收集本院1999年4月至2009年9月诊断的46例符合组织学三联征形态改变的石蜡包埋淋巴结组织,采用半嵌套式PCR方法 对提取DNA进行弓形虫基因组的片段扩增;另选取30例组织中可见三联征中的二个或一个特征的病例作为对照.结果 组织学三联征组PCR阳性率为76.1%(35/46),对照组阳性率仅为10.0%(3/30,P<0.01);组织学三联征诊断弓形虫淋巴结炎的灵敏度为92.1%(35/38),特异度为71.1%(27/38);PCR法的阳性预测值为76.1%(35/46),阴性预测值为90.0%(27/30).结论 经典的组织学三联征对于诊断弓形虫淋巴结炎的特异性很强,但敏感性较低,且易漏诊部分非典型病例.在组织形态改变的基础上结合半嵌套式PCR对刚地弓形虫基因片段的检测可大大提高检出敏感性和准确性.     

组织学三联征和聚合酶链反应在弓形虫淋巴结炎病理诊断中的应用

目的 经典的组织学三联征诊断法和荧光原位杂交(PCR)法在提高石蜡包埋组织弓形虫淋巴结炎确诊率中的价值.方法 收集本院1999年4月至2009年9月诊断的46例符合组织学三联征形态改变的石蜡包埋淋巴结组织,采用半嵌套式PCR方法 对提取DNA进行弓形虫基因组的片段扩增;另选取30例组织中可见三联征中的二个或一个特征的病例作为对照.结果 组织学三联征组PCR阳性率为76.1%(35/46),对照组阳性率仅为10.0%(3/30,P<0.01);组织学三联征诊断弓形虫淋巴结炎的灵敏度为92.1%(35/38),特异度为71.1%(27/38);PCR法的阳性预测值为76.1%(35/46),阴性预测值为90.0%(27/30).结论 经典的组织学三联征对于诊断弓形虫淋巴结炎的特异性很强,但敏感性较低,且易漏诊部分非典型病例.在组织形态改变的基础上结合半嵌套式PCR对刚地弓形虫基因片段的检测可大大提高检出敏感性和准确性.     

Utility of the polymerase chain reaction in the diagnosis of tuberculous meningitis

Due to inconsistent clinical presentations and the lack of a rapid, sensitive and specific test, tuberculous meningitis (TBM) is particularly difficult to diagnose. The present study was carried out to determine the utility of the polymerase chain reaction (PCR) using INS primers in the diagnosis of TBM and to compare the efficacy of two different DNA extraction protocols. Fifty-seven cerebrospinal fluid (CSF) samples from suspected cases of meningitis -- 30 definitive/possible TBM and 27 non-TBM -- were processed for microscopy, culture and PCR. Results of computer tomographic (CT) scan findings were noted. The results of smear, culture and PCR were compared using culture and/or clinical response to treatment as the gold standard. The sensitivity of microscopy, culture, CT scan and PCR was 3.3%, 26.7%, 60.0% and 66.7%, respectively. PCR following QIAmp DNA extraction had a sensitivity of 66.7% compared to PCR following a DNA extraction protocol based on the use of cetyl trimethyl ammonium bromide (CTAB) (50%). PCR was positive in all culture-positive CSF samples using either extraction method. PCR is a rapid and sensitive technique; above all, it can diagnose tuberculous meningitis at a very early stage.     

Rapid diagnosis of tuberculous lymphadenitis by amplification of mycobacterial DNA]

In patients with cervical lymph node swelling, prompt differential diagnosis of tuberculosis leads to an appropriate treatment. A method based on DNA amplification and hybridization for the rapid detection of Mycobacterium tuberculosis was used for demonstrating its specific DNA in cervical lymph nodes biopsied from seven patients. The DNA specific for Mycobacterium tuberculosis was detected in 5 patients. Four of them were histologically diagnosed as tuberculous lymphadenitis. In another case with necrotizing lesions but without granulomatous reactions, culture method subsequently revealed a positive result. In other 2 cases, the diagnosis of tuberculosis was promptly excluded by both this method and conventional methods (histologic diagnosis/direct microscopy). The rapid detection of Mycobacterium tuberculosis by amplification of mycobacterial DNA is helpful particularly in differential diagnosis of cervical lymphadenopathy.     

DNA analysis by polymerase chain reaction]

Polymerase chain reaction (PCR) is a technique to amplify only a specific segment of DNA without using a plasmid or a phage vector. It is a powerful tool for genetic analysis of various diseases including inherited and viral diseases, and is now being applied to clinical diagnosis. Here, presented are several methods using PCR mainly for diagnosis of hemoglobinopathy which we have been engaged in. Some other diseases are also included.     

Specificity of the histopathological triad for the diagnosis of toxoplasmic lymphadenitis: polymerase chain reaction study

Toxoplasmosis is a common cause of lymphadenopathy, but toxoplasmic cysts are not usually found in histological sections used for establishing diagnosis, except on extremely rare occasions. The histopathological triad of florid reactive follicular hyperplasia, clusters of epithelioid histiocytes, and focal sinusoidal distention by monocytoid B cells has been considered to be diagnostic of toxoplasmic lymphadenitis, but the validity of the histopathological triad is based indirectly on serological correlation only. The demonstration of Toxoplasma gondii DNA in lymph nodes displaying the histopathological triad will indicate the validity of the histopathological triad as the criterion for the histopathological diagnosis of toxoplasmic lymphadenitis. We used frozen tissues of 12 lymph nodes with the histopathological triad and tissues of 27 lymph nodes from patients with various other conditions (including 13 cases of follicular lymphoid hyperplasia, FLH; three cases of dermatopathic lymphadenopathy, DPL; two cases of plasmacytosis; two cases of Castleman's disease; two cases of metastatic adenocarcinoma; and five cases of lymphoma) to detect T. gondii DNA by polymerase chain reaction. Ten out of 12 lymph nodes with the triad and six out of 27 lymph nodes without the triad were positive for T. gondii DNA. Thus, the sensitivity of the triad was 62.5% (10/16) and the specificity was 91.3% (21/23). The predictive value of positive tests was 83.3% (10/12) and the predictive value of negative tests was 77.7% (21/27). The six cases positive for T. gondii DNA without the triad were four cases of FLH, one case of DPL, and one case of plasmacytosis. None of the neoplastic diseases was positive. The false positive and negative cases could be due to sampling problems or past T. gondii infection. The results confirm that the histopathological triad is highly specific for the diagnosis of toxoplasmic lymphadenitis and can be used confidently.     

Detection and diagnosis of parapoxvirus by the polymerase chain reaction

The genus Parapoxvirus includes four members, bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), orf virus (ORFV) and parapoxvirus of red deer in New Zealand (PVNZ). A set of primers for polymerase chain reaction (PCR) was designed to detect viral DNA from cells infected with each of the four parapoxviruses. The set of primers resulted in the amplification of appropriately sized products from cells infected with BPSV, PCPV, ORFV and PVNZ, respectively. The PCR method was applied for the detection of seven field isolates of parapoxvirus from cattle, sheep and free-ranging wild Japanese serows. The expected size of DNA was amplified from cells infected with each of the seven isolates. No specific PCR products were detected from vaccinia virus-, fowlpox virus- and mock-infected cells. Moreover, by a semi-nested PCR with an inner primer and Southern blot analysis, viral DNA was detected from lesions of clinically affected cattle, sheep and Japanese serows. These results suggested that the PCR method used in this study was specific for the detection of parapoxviruses and thus useful for diagnosis of parapoxvirus infections, especially in discrimination from diseases with similar clinical symptoms.     

Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction]

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.