Kaposi肉瘤中人疱疹病毒8的检测及其临床病理学意义

目的 了解Kaposi肉瘤与人疱疹病毒8（HHV8）或Kaposi肉瘤相关病毒的关系;建立适合于临床病理诊断的原位杂交方法。方法 采用地高辛苦标记的单链HHV8 DNA探针,辅以增强探测敏感性的信号扩增系统（tyramide signal amplification）对14例甲醛固定石蜡包埋的Kaposi肉瘤组织标本进行了HHV8原位检测,为了比较,同时也采用了聚合酶链反应（PCR）检测技术。结果 用原位杂交和PCR两种方法共发现10例（71%）Kaposi肉瘤组织中存在HHV8感染。其中8例在两种技术检测均呈阳性。HHV8阳性信号见于肿瘤梭形细胞和内皮细胞,也见于各期病变（早中期的斑片和斑块病变,晚期的结节病变）,以及原发,复发和（或）转移病变。所有阴性对照在两种方法中均呈阴性。结论 Kaposi肉瘤与HHV8存在密切关系;这种地高辛标记单链DNA探针,辅以信号扩增系统的原位杂交技术检测该病毒具有用于临床病理诊断的价值。     

原位PCR技术在检测鼻咽癌组织中bcl-2基因重排的应用

为使原位聚合酶链反应（ISPCR）技术更具敏感性及特异性,我们建立了半套式原位PCR技术,并应用该技术对鼻咽癌组织中bcl-2基因重排进行检测,并与分子原位杂交进行比较,获得满意效果。     

聚合酶链反应结合反向线点杂交技术在检测及鉴定真菌性鼻窦炎常见致病曲霉菌中的应用

目的 探讨聚合酶链反应结合反向线点杂交(PCR/RLB)技术在检测和鉴定真菌性鼻窦炎(FS)常见致病曲霉菌中应用的可行性.方法 收集首都医科大学附属北京同仁医院2009年5月至2010年2月住院手术的FS患者活检石蜡包埋组织26例和活检新鲜组织8例,全部病例进行了病理学真菌检查、真菌培养及真菌ITS2区测序;提取各标本中真菌的DNA,用真菌特异性引物行PCR扩增,针对真菌的ITS2区设计曲霉菌种特异性探针,用5根曲霉菌种特异性探针与PCR产物进行反向线点杂交;将PCR/RLB与ITS2区测序、真菌培养及病理学检查的结果进行比较.阳性对照为5株曲霉菌株,阴性对照为真菌培养为非曲霉的石蜡包埋组织.结果 活检标本病理学检查均可见菌丝;真菌培养14例阳性(41.2％);测序16例(47.1％)得到结果;PCR/RLB鉴定34例均得出鉴定结果:黄曲霉14例,烟曲霉10例,黑曲霉4例,构巢曲霉1例,黄曲霉和烟曲霉同时阳性3例,烟曲霉与黑曲霉同时阳性2例.结论 PCR/RLB技术可以对FS常见致病曲霉菌进行检测及鉴定,与病理组织学检查、真菌培养及DNA测序方法相比较,具有经济省时、敏感性高、特异性强、高通量等优势.     

石蜡包埋肝组织中丙型肝炎病毒的原位PCR检测

以丙型肝炎病毒（HCV）为研究对象探讨原位PCR这一新技术。用原位PCR和原位杂交检测石蜡包理肝组织中的HCV。7例肝硬变及8例肝细胞癌痛旁组织HCV的检出率分别为86％（6／7例）和50％（4／8例），明显高于同组原位杂交的42％（3／7例）和12．5％（1／8例），并且显示了良好的特异性。研究发现HCV主要分布于肝细胞浆中，并且在胆小管上皮细胞及单核细胞中也出现阳性信号。我们的研究表明原位PCR是一项敏感、特异的方法，它的广泛应用不仅可以提高丙型肝炎的诊断率，同时有助于其分子病理学的研究。     

介绍一种半套式原位PCR技术

原位聚合酶链反应（原位PCR）是具有聚合酶链反应（PCR）技术的高敏感性和原位来交（IS）肘原位检测能力的新方法。但目前常见的间接式原位PCR技术常因PCR反应过程的非特异扩增而伴有假阳性结果出现,对此我们建立了一种高度特异性的卡套式原位PCR法,并与ISH方法进行了比较。1材料与方法利例石蜡包埋鼻咽癌组织,切片知m,贴于涂有防脱片胶的载玻片L6OoC24h。半套式PCR一对半引物及生物素标记寡核苦酸探针l’l由上海生物工程研究所合成。引物为hcf－2基因主要断裂区外引物A！、内引物AZ及IgV。基因引物JH。半套式原位PCR步…     

应用实时荧光PCR技术检测HBV YMDD变异

目的　应用YMDD变异荧光PCR检测试剂盒检测乙型肝炎病毒 (hepatitisBvirus,HBV)聚合酶基因区的酪氨酸 蛋氨酸 天门冬氨酸 天门冬氨酸基序 (tyrosine methionine asparticacid asparticacid ,YMDD)变异 ,希望能够替代目前使用的测序方法。方法　对 4 7例血清标本使用荧光PCR技术检测YMDD变异 ,其YMDD变异结果与DNA测序结果进行比较。结果　荧光PCR方法测得YMDD野生株 2 8例 ,变异株 19例 ;与DNA测序结果完全一致 ,符合率为 10 0 %。结论　荧光PCR技术检测YMDD变异具有方便、快速、准确的特点。     

利用聚合酶链反应技术（PCR）测定早孕绒毛组织中巨细胞病毒DNA的研究

以人工合成的人巨细胞病毒DNA片断为引物,利用聚合酶链反应技术(PCR)检查早孕绒毛标本中人巨细胞病毒感染,对50例人工流产的早孕绒毛组织标本进行PCR基因扩增,40例绒毛标本中捡出HCMV。与同一绒毛标本的免疫组织化学染色结果相比较,PCR技术为早期、快速进行产前诊断,决定处理原则提供了一个新的检测手段。     

藏族株TT病毒的分子生物学研究

目的 初步探索青海少数民族地区输血后肝炎病毒（TTV）感染的分子流行病学和TTV DNA部分核苷酸片段序列的分析。方法 采用ELISA聚合酶链反应（PCR）技术,检测藏族、蒙古族、土族人群TTV的感染性,TTV DNA应用荧光法测序分析部分核苷酸片段。结果 ELISA检测74例藏族TTV阳性率14．9％;57例蒙古族TTV阳性率1．9％;土族未检测到阳性。PCR检测13例TTV阳性血清,DNA阳性率53．8％;藏族TTV DNA部分核苷酸测序,对照BDH1．Shanxi13.Gla序列,同源性达90％以上。结论 TTV在青海少数民族人群间有一定的感染性,不同区域的民族感染率有显著差异。藏族株TTV DNA片段对照分析同源性为90％以上,属Gla亚型。提示民族间感染有输入性特征。     

无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

肝细胞癌病理学研究的新进展

1 原位PCR检测石蜡包埋肝组织中丙型肝炎病毒 HCV是单股正链RNA病毒,Negro等用原位杂交研究发现HCV共存于人肝组织细胞核及细胞质中,但主要位于胞质中。原位PCR检测肝组织HCV感染的敏感性比原位杂交高40％-50％,血清HCV阳性患者的肝组织检出率高达86％”。尤其对HBsAg阴性的癌旁组织进行检测可使50％原因不明的肝癌得到诊断。存档多年的蜡块也可进行检测。     

原位PCR技术检测石蜡包埋脑组织中人巨细胞病毒DNA

应用原位聚合酶链反应（ＩＳＰＣＲ）技术检测了２５例尸检畸形胎儿石蜡包埋脑组织中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ，并与普通ＰＣＲ及原位杂交（ＩＳＨ）进行了比较。ＩＳＰＣＲ、ＰＣＲ及ＩＳＨ检测阳性率分别为４４％，３６％及２０％。与ＩＳＨ相比较，ＩＳＰＣＲ不仅检出阳性率高，而且信号强度增强。研究结果提示，ＩＳ－ＰＣＲ是诊断ＨＣＭＶ感染的快速、敏感、特异的实用方法。     

In situ polymerase chain reaction detection of viral DNA, single-copy genes, and gene rearrangements in cell suspensions and cytospins.

The study of low-copy viral or genomic DNA sequences by in situ hybridization (ISH) is often limited by sensitivity. Using the polymerase chain reaction (PCR) for the amplification of target DNA sequences in fixed cells [in situ PCR] (ISPCR) before ISH, we have been able to greatly improve the sensitivity of ISH. Viral DNA present in low copy number, single-copy genes, as well as immunoglobulin gene rearrangements (VH3 family genes), were successfully amplified in cells in suspension or on glass slides (cytospins). Single primer pairs were used in the in situ amplification step and 35S- or digoxigenin-11-dUTP-labeled region specific oligonucleotide probes were used for detection of amplificants by ISH. Artifacts, presumably resulting from leakage of in situ amplificants out of cells, may be a significant problem in selected instances. By optimal fixation and permeabilization of cells, limiting PCR cycle number, amplification of long DNA sequences, and/or incorporation of biotinylated dNTPs to produce bulkier amplificants together with washing of cells after ISPCR, diffusion artifacts were significantly reduced. Probe hybridization to single-stranded long PCR fragments or messenger RNA were excluded as a source for false-positive ISPCR results. The techniques reported dramatically increase the sensitivity of ISH in the detection of low-copy viral infection as well as in the study of gene rearrangements, and provide unique opportunities to study chromosomal translocations and point mutations at the cellular level.     

In situ hybridisation and in situ polymerase chain reaction detection of parvovirus B19 DNA within cells

Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.     

原位检测新基因SNC66在胃癌组织与正常胃粘膜中的表达

目的: 观察新基因SNC66在胃癌组织中的表达水平,分析SNC66表达水平与胃癌发生的相关性。 方法:以β-actin为对照,对20例胃癌组织及其配对正常胃粘膜的石蜡切片,进行原位cRNA/mRNA分子杂交和原位RT-PCR扩增。 结果:两种检测方法表明,SNC66在正常胃粘膜组织中都呈强阳性表达。而在胃癌组织中,原位分子杂交检测表明,20例标本SNC66不表达和弱阳性表达各8例,强阳性表达4例。原位RT-PCR检测表明,当循环数为10时,此20例胃癌标本中不表达、弱阳性和呈强阳性表达数分别为6,9和5;而当循环数为25时,则4例不表达,16例呈强阳性表达。 结论:SNC66基因在胃癌组织中存在明显的表达降低和表达缺陷。SNC66可以作为一个胃癌负相关基因加以进一步研究。     

原位PCR检测霍乱弧菌ctxAB基因

目的　为监测环境中的霍乱弧菌 ,建立原位PCR(insituPCR ,ISPCR)检测方法。方法纯培养的霍乱弧菌为实验材料 ,霍乱毒素基因 (ctxAB)为靶序列。 4 %多聚甲醛固定细胞 ,1mg/ml溶菌酶消化 2 0min ,PCR反应体系中加入 2 .5 %甘油 ,以荧光素标记的引物为标示物 ,在液相环境下 ,进行靶序列的ISPCR。结果　经蓝光激发 ,荧光显微镜下有扩增产物的细菌发出明亮的绿色荧光 ,阴性对照则无荧光。结论　由于ISPCR既能检测靶序列 ,又保护了细菌形态 ,可以在检测中省去细菌培养的过程 ,因此 ,为快速而准确的检测环境中霍乱弧菌提供了一种新方法     

Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization

The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as weli as in sltu hybridizetion (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-ernbedded urinary bladder cancem, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16-or 18-positive signals in ail except one of the tumor samples. However, the FCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffinembedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers.     

Detection of Epstein-Barr virus genome within thymic epithelial tumours in Taiwanese patients by nested PCR,PCR in situ hybridization,and RNA in situ hybridization

Epstein-Barr virus (EBV) is known to be associated with a variety of tumours, including Burkitt's lymphoma, nasopharyngeal carcinoma, and some carcinomas of other organs with similar lymphoepithelioma-like features. The association between EBV and thymic epithelial tumours is inconclusive, as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. This study examined 78 thymomas and 21 thymic carcinomas in Taiwanese patients, to detect the viral genome at both DNA and RNA levels. The tissue blocks were first screened by nested polymerase chain reaction (PCR) targeting on the first tandem internal repeats. The positive cases were further submitted for viral localization by in situ PCR insitu hybridization (ISH) and Epstein-Barr-encoded RNA-1 (EBER-1) ISH. None of the thymomas showed a detectable EBV genome. Eight thymic carcinomas were positive for EBV by nested PCR, of which six displayed nuclear signals within the tumour cells by in situ PCR ISH and/or RNA ISH, one displayed signals within the lymphocytes, and one showed no discernible in situ signals. Most of them exhibited a lymphoepithelioma-like morphology. These results show that nested PCR is a sensitive method for screening the EBV genome in thymic epithelial tumours. In situ PCR ISH is reliable for localization of the virus, in addition to EBER-1 RNA ISH. Thymomas are not related to EBV, even in this endemic area. Thymic carcinomas, especially the lymphoepithelioma-like thymic carcinomas, are more often associated with the virus.     

Human papillomavirus infection is not associated with bronchial carcinoma: evaluation by in situ hybridisation and the polymerase chain reaction

While a strong association between human papillomaviruses (HPVs) and squamous cell cancers of the female genital tract is known to exist, there is substantial controversy regarding the relationship of HPV with other non-genital carcinomas. Recently there have been some reports focusing on a possible association of HPVs with bronchial carcinomas. These studies mostly used either in situhybridization (ISH) or the polymerase chain reaction (PCR). In view of these reports, 32 squamous cell carcinomas (SCCs) and six small cell carcinomas of the bronchus were examined for the presence of HPV DNA by both techniques: ISH using ³⁵S-labelled, type-specific probes (HPV 6, 11, 16, 18), and PCR with consensus primers coding for more than 25 different HPV subtypes performed on formalin-fixed, paraffin-embedded material. None of the 38 bronchial carcinomas analysed was positive for HPV DNA, either by ISH or by PCR. On the other hand, additionally examined specimens of 15 cervical carcinomas were positive for HPV 16 DNA in at least three cases by ISH (20 per cent) and in 12 cases by PCR (80 per cent). We conclude that common HPV types do not play an important role in the pathogenesis of bronchial carcinoma. © 1997 John Wiley & Sons, Ltd.