地高辛精标记RNA探针原位杂交的简便方法

用地高辛精标记ＴＨ基因合成ＲＮＡ探针，在冰冻组织切片上进行原位杂交，通过碱性磷酸酶催化的呈色反应，检测在基因治疗帕金森病大鼠动物模型研究中酪氨酸羟化酶基因在脑内的表达。实验结果表明，简化原位杂交方法可适应于任何实验室。     

一种生物素标记探针DNA—RNA原位杂交方法

一种生物素标记探针ＤＮＡ—ＲＮＡ原位杂交方法王建明，李凌，沈贵华，崔惠云，李申德我们采用国产生物素光标记试剂盒标记探针，用于细胞涂片，冰冻切片及石蜡切片的原位杂交，检测细胞中目的基因的转录产生水平。一、材料和方法１．标本：人大肠肿瘤手术标本，冰冻切片...     

地高辛素标记探针原位杂交法检测肝组织中丙型肝炎…

以国外引进的含丙型肝炎病毒ｃＤＮＡ的质粒ＤＮＡ为模板，应用地高辛素作为标记物，经聚合酶链反应制备探针，建立原位杂交方法，对４１例石蜡包埋肝组织进行ＨＣＶ，ＲＮＡ测定。探针标记的ＨＣＶ基因片段位于５‘非编码区，其长度为２２１ｂｐ。结果：抗ＨＣＶ阳性组的ＨＣＶ ＲＮＡ阳性检出率为５６．５％，抗－ＨＣＶ阴性组为１６．７％。     

RNA原位杂交、荧光原位杂交和免疫组织化学检测宫颈癌PD-L1和galectin-9比较研究

目的 分析不同检测方法检测宫颈癌组织程序性死亡配体-1 (programmed death ligand 1,PD-L1)和T细胞免疫球蛋白黏蛋白分子-3(T-cell immunoglobulin mucin 3,TIM-3)配体半乳糖凝集素-9(galectin-9)的一致性.方法 选择宫颈癌患者100例,分别采用...     

卡氏肺孢子虫病的诊断及其进展

卡氏肺孢子虫病诊断方面的研究表明，辅助诊断方法缺乏特异性，在确定病人是否有肺感染，感染的程度及疗效的考核上都有一定作用。但ＰＣ感染的最后确诊主要靠病原体检测。取材方法：对于肺感染者应以导痰为首选，其次是ＢＡＬ和ＴＢＬＢ。儿童可采用经皮穿刺肺吸引的方法取材。在前述方法无法实施的紧急情况下，才考虑开胸肺活检。肺外感染者可在病变局部穿刺取材。制片法：可用穿刺物或肺组织等做直接涂片，印片，或悬液滴片等简捷方法，也可采用组织切片。染色方法：常用Ｇｉｅｍｓａ、初检、ＴＢＯ或ＧＭＳ法进一步确诊。ＩＦ可提高检出率。ＰＣＲ技术检测孢子虫病更敏感和特异，有操作简便，易于推广应用等优点。     

RNA原位杂交技术难点及针对措施

利用互补RNA为探针进行原位杂交是分析组织或细胞内RNA分布行之有效的方法,通过对RNA分布的研究可以了解特定基因的表达情况。用寡核苷酸探针检测组织切片中相关的mRNA是较为常见的RNA原位杂交。该探针方法简便、探针较短,组织穿透性好,有较高的灵敏度。但是过程较长,操作繁琐,     

mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。     

系统性红斑狼疮并发肺孢子虫肺炎的临床病例报告

目的探讨系统性红斑狼疮并发肺孢子虫肺炎（PCP）的诊断和防治措施。方法研究1例系统性红斑狼疮并发肺孢子虫肺炎患者的临床表现、实验室检查结果、影像学资料以及诊断和治疗的方法。结果患者出现发热、干咳、无痰、胸闷、伴阵发性呼吸困难等症状,血氧饱和度下降,胸部CT显示两肺部毛玻璃状改变,肺泡灌洗液找到肺孢子虫。给予复方新诺明、卡泊芬净、调整免疫抑制剂及其它支持对症治疗,痊愈出院。结论免疫功能抑制患者有发热、干咳、呼吸困难等临床症状,应考虑PCP并及时做病原学检查。     

肺鳞癌人乳头状瘤病毒感染的原位杂交检测和观察

经多重多聚酶链反应，检测４９例肺鳞癌中发现７例人乳头瘤病毒阳性的肿瘤组织，采用生物素标记ＨＰＶＤＮＡ探针，进行原位杂交检测，结果发现在５例肿瘤组织中显示ＨＰＶＤＮＡ阳性信号，其中ＨＰＶ１１型阳性３例；ＨＰＶ１６例阳性１例；１例为ＨＰＶ１１例和１６型均阳性。原杂交ＨＰＶＤＮＡ阳性信号，大多位于凹空样肿瘤细胞或低分化鳞癌细胞的核内，分子生物学研究表明ＨＰＶ感染可能与部分鳞有关。     

环介导等温扩增技术检测卡氏肺孢子虫的研究

目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论 检测Pc的LAMP方法敏感、特异及简便.     

Detection by in situ hybridization of HIV and c-myc RNA in tumour cells of AIDS-related B-cell lymphomas

The incidence of acquired immune deficiency syndrome (AIDS)-related malignant lymphoma has increased since the disease was first described, but its pathogenesis is still not understood. There have been numerous molecular studies addressing the clonality of these proliferations, the presence of Epstein-Barr virus genome in the tumour cells, and rearrangements of the c-myc oncogene. However, very few in situ hybridization studies have been carried out. We analysed 24 cases of high-grade B-cell malignant lymphomas and two cases of polymorphic B-cell proliferation associated with human immunodeficiency virus (HIV) infection. Human immunodeficiency virus ribonucleic acids were detected in some of the tumour cells in 19 of the 24 cases of malignant lymphomas and in both cases of polymorphic B-cell proliferation, with the in situ hybridization technique and using a specific tritiated copy deoxyribonucleic acid probe. With the same technique, c-myc ribonucleic acid was detected in most of the tumour cells from all the 21 cases of malignant lymphomas tested but not in the polymorphic B-cell proliferation.     

Pneumocystis carinii in FNA of the thyroid.

We report the diagnosis of Pneumocystis carinii (PC) in a fine-needle aspirate (FNA) from the thyroid of a human immunodeficiency virus infected (HIV+) male receiving aerosolized pentamidine as prophylaxis for Pneumocystis carinii pneumonia (PCP). The clinical diagnosis prior to FNA was multinodular goiter. The patient did not have pulmonary symptoms nor previous diagnosis of PCP at the time of the aspirate diagnosis. Recently, extrapulmonary Pneumocystis carinii (EPC) has been reported with increasing frequency in HIV+ patients receiving prophylactic aerosolized pentamidine. Awareness of extrapulmonary presentations of Pneumocystis carinii infection is a prerequisite for accurate cytologic diagnosis.     

Observations favouring Pneumocystis carinii pneumonia as a primary infection: a monoclonal antibody study on paraffin sections

Pneumocystis carinii pneumonia is characteristic of immunodeficiency and the organism is probably acquired during early childhood. Since infection is only manifest in the lungs, it has been presumed that the organism lies dormant in these tissues following the primary infection. Conventional staining procedures have, however, failed in the absence of pneumonia to demonstrate consistently any forms of Pneumocystis carinii. To study this problem further, lung sections and hilar lymph nodes from immunodepressed adults with and without Pneumocystis carinii pneumonia as well as lung sections from presumed immunocompetent patients were examined for the cyst and trophozoite forms of Pneumocystis carinii using a monoclonal antibody. The organism was only identified in areas of pneumonia, and the source of the organism in these patients may therefore be a new infection with a different human subtype and not, as previously thought, reactivation of a primary infection.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

Detection of rhinovirus RNA in middle turbinate of patients with common colds by in situ hybridization

Human rhinovirus 14 RNA was determined by in situ hybridization from middle turbinate biopsies in 32 patients with diagnosed common colds and in five control individuals. Twenty-two (69%) biopsies from common colds patients but none of the five control biopsies showed reactivity for human rhinovirus 14 antisense probe. The signal was detected both in the respiratory epithelium and in mucosal inflammatory cells. In situ hybridization of the middle turbinate tissue yielded more positive results than RT-PCR (47%) or virus culture (34%) assayed from nasopharyngeal aspirates, but no statistical significant differences were observed (P = 0.265, P = 0.425, respectively). The results indicated that in situ hybridization procedure was slightly more sensitive than PCR assays and classical culture for the detection of human rhinovirus infection of upper respiratory tract. However, in situ hybridization procedure appeared to be an interesting methodology to investigate the physiopathology of respiratory tract infection by rhinoviruses.     

A search for Pneumocystis carinii in post-mortem lungs by DNA amplification.

DNA amplification of specific sequences and subsequent oligonucleotide hybridization were used to search for Pneumocystis carinii in post-mortem lung samplings from non-immunosuppressed individuals ranging from 15 to 70 years of age. No P. carinii-specific DNA was detected in 45 DNA amplification reactions from 15 lungs.     

Detection of Epstein–Barr virus small RNAs in routine paraffin sections using non-isotopic RNA/RNA in situ hybridization

The Epstein–Barr virus (EBV) is associated with an increasing range of reactive and neoplastic lesions. There is a need for a sensitive and specific method for detecting latent EBV in routine histological sections. We report the use of a highly sensitive paraffin section RNA/RNA in situ hybridization (ISH) technique using digoxigenin-labelled antisense riboprobes for demonstrating EBV encoded-small RNAs (EBERs), EBV gene products that are transcribed in abundance during latent EBV infection. We applied EBER-ISH to 846 paraffin embedded specimens, including cases of reactive lymphoid hyperplasia ( n = 28), infectious mononucleosis (16), Burkitt's lymphoma (44), immunodeficiency-associated lymphomas in transplant recipients (9) and AIDS patients (128), Hodgkin's disease (130), CD30 antigen positive lymphomas (106), peripheral T-cell lymphomas (104), sporadic B-cell non-Hodgkin's lymphomas (162), undifferentiated nasopharyngeal carcinoma (86), salivary gland lymphoepithelioma (11), and oral hairy leukoplakia (5). Strong, reproducible EBER staining was seen in EBV latently infected cells in archival surgical biopsy and autopsy specimens. EBER-ISH is specific, has a sensitivity comparable to that of the polymerase chain reaction, and is now the method of choice for the in situ detection of latent EBV infection.