用PCR标记核酸探针原位检测尖锐湿疣中HPV的结果观察

用ＰＣＲ标记核酸探针原位检测尖锐湿疣中ＨＰＶ的结果观察邵建永，吴秋良，侯景辉，周家杰，陈火胜中山医科大学肿瘤医院病理科（邮政编码５１００６０）用ＰＣＲ标记ＨＰＶＤＮＡ探针，对２０例尖锐湿疣（ＣＡ）和乳头状瘤（Ｐａｐｉｌｌｏｍａ，Ｐａｐ）做原位杂交（Ｉ...     

029尖锐湿疣患者阴毛中HPV6型和11型的检测

尖锐湿疣由人乳头瘤病毒 (ＨＰＶ) 6型及 11型等感染引起。治疗较为困难 ,而且容易复发。其复发率高的原因尚不清楚 ,推测可能与再次感染或潜伏的ＨＰＶ被重新激活有关。作者采用ＰＣＲ检测从尖锐湿疣患者拔出的阴毛及眉毛上的ＨＰＶＤＮＡ ,探讨了毛囊处潜伏的ＨＰＶ与尖锐湿疣复发的关系。2 5例尖锐湿疣患者 ,经同意后用镊子拔取耻骨部、肛周及眉毛部 3处的毛发各 5～ 8根 ,要求根部附带毛囊组织。标本贮存于 - 70℃ ,用异硫氰酸胍 -硅藻土方法提取ＤＮＡ。应用ＣＰⅠ ＣＰⅡｇＰＣＲ法检测标本中的ＨＰＶＤＮＡ ,并进行序列分析 ,测定Ｈ…     

尖锐湿疣组织学诊断与病原学检测的探讨

根据人类乳头瘤病毒感染外阴皮肤粘膜后的组织学改变性特征诊断尖锐湿疣，抽取３１例行病毒核壳抗原免疫组化染色和ＰＣＲ检测ＨＰＶＤＮＡ。结果：ＨＰＶ－Ａｇ的检出率为８３．８７％，ＨＰＶＤＮＡ的检出率为１００％（３１／３１）。     

尖锐湿疣新鲜组织HPV的聚合酶链反应检测与斑点杂交比较的研究

人乳头瘤病毒（ＨＰＶ）６／１１型是尖锐湿疣的主要病原体［１～３］。根据文献报导，以往检测方法很多，均无特异性，故从病原体的角度进行检测，具有重要的意义。我们将临床诊断力尖锐湿疣的患者７７例新鲜组织标本，用ＰＣＲ法检测ＨＰＶ６／１１型ＤＮＡ，并与斑点杂交法进行比较，现总结如下。一、病例选择ＣＡ患者共７７例，男４０例，女３７例，年龄１８～５６岁，平均３７岁，已婚４５例。病程最长者１年，最短１５天。发病部位：大小阴唇、阴道口、尿道口、龟头、冠状沟、包皮、肛ｌ’１周围等处。二、方法（一）模板ＤＮＡ提取：…     

人类乳头瘤病毒16全基因在体外培养的正常人角质形成细胞 …

目的 利用含有人类乳头瘤病毒（ＨＰＶ）１６全基因的质粒转染正常人角质形成细胞，观察ＨＰＶ１６ｍＲＮＡ在转染细胞的表达。方法 用ＦｕＧＥＮＥ＾ＴＭ６转染试剂，将携带ＨＰＶ１６全基因的质粒ｐＳＶ２－ｎｅｏ／１６转染体外培养的正常人角质形成细胞，在转染后２４ｈ提取细胞总ＲＮＡ和ＤＮＡ，进行ＲＴ－ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹分析，结果 ２４ｈ后，ＲＴ－ＰＣＲ成功地扩增出１１０ｂｐ的片段，转染细胞中已出现Ｈ     

聚合酶链反应检测生殖器疣与癌中人乳头瘤病毒

一种新建立的总引物介导的聚合酶链反应（ＰＣＲ）可用以检测多种人乳头瘤病毒（ＨＰＶ）基因型别。结果： ＨＰＶ ＤＮＡ片段检出率：生殖器疣９８％（９８／１００）、阴茎表皮肉瘤Ⅲ级８０％（１０／１２）、宫颈上皮内启 ８０％（１２／１５）、阴茎鳞癌８０％（１６／２０）及宫颈鳞癌９０％（２７／３０）；其中，９５％（９３／９８）的生殖器疣中检出ⅢＨＰＶ６、１１，而生殖器癌中ＨＰＶ１６伴同率为８８％（３８／４３）。上述结果提示； ＰＣＲ技术系检测病因疑为ＨＰＶ的非典型增生及癌中ＨＰＶ病原的有效方法，ＨＰＶ６、１１和ＨＰＶ１６分别为本地区生殖器ＨＰＶ感染良、恶性病变中之流行型别。     

聚合酶链反应和斑点杂交方法检测人乳头瘤病毒DNA

目前用于检测ＨＰＶ感染的方法很多，我们对聚合酶链反应（ＰＣＲ）和斑点杂交方法进行了比较研究。一、临床资料１７例尖锐湿疣病例均来自１９９０～１９９１年我院皮肤科和妇科外阴门诊，５％醋酸白试验均阳性。所有病例均取病损活检，经病理诊断为尖锐湿疣。二、方法（一）ＰＣＲ１．寡核着酸引物的设计：根据已公布的病毒基因组序列设计寡核着酸引物序列，扩增４型ＨＰＶ的高保守区Ｅ６区［‘·’］。由于尖锐湿疣主要是ＨＰＶ６、１１型引起，ＨＰＶ６和１１型的基因组Ｅ６区有同源序列，故将ＨＰＶ６、１１型设计了一对共同引物，引物…     

PCR检测164例STD患者5种常见病原体结果

ＰＣＲ检测１６４例ＳＴＤ患者５种常见病原体结果黄干军近年来我们用聚合酶链反应（ＰＣＲ）对１６４例性传播疾病（ＳＴＤ）患者进行了淋球菌（ＧＣ）、沙眼衣原体（ＣＴ）、解脲支原体（ＵＵ）、人类乳头瘤病毒（ＨＰＶ）和单纯疱疹病毒（ＨＳＶ）的ＤＮＡ检测，现报告...     

应用PCR技术检测人乳头瘤病毒DNA

从ＨＰＶ基因组Ｌ１区段选择了大多数亚型均具有的保守序列，借助计算机检索验证，设计出一对适用 于检测１７种ＨＰＶ亚型的引物。并应用该引物对５１例病理诊断为尖锐湿（ＣＡ）的标本进行了ＰＣＲ扩增检测。结果表明，其枵次性完成对多种ＨＰＶ亚型感染的检测，检测覆盖而宽，漏检率低，对ＣＡ的论断确有较好的应用价值。     

CO2激光治疗尖锐湿疣烟尘中HPV DNA的检测

为了明确ＣＯ２激光治疗尖锐湿疣过程中，烟尘中是有ＨＰＶ存在，采用ＰＣＲ方法对尖锐湿疣患者治疗时烟尘标本和组织块标本进行ＨＰＶ６和１１型ＤＮＡ检测。结果：２１例组织块中，１９例阳性；从距激光烧灼部位５ｃｍ（２１例）和５０ｃｍ（１２例）处收集的烟尘标本中，无１是性。实验中未发现粝尘中及不同的吸附材料（无纺布、纱布和擦镜纸）存在对ＨＰＶ检测有抑制作用的物质。本实验的结果提示：在用大功率ＣＯ２激光治疗疣时     

巢式PCR方法检测非生殖器部位Bowen病HPV DNA

目的 建立一种巢式ＰＣＲ方法,探讨HPV DNA在非生殖器部位Bowen病中的检出率。方法 巢式PCR方法,用5对不同引物包括CN1FR、CN2FR、CN3FR、CN4FR以及CN5FR进行扩增。结果 通过ClustalX软件,将所设计的每对引物与已知HPV亚型的碱基序列逐一相比较,得知改良设计的引物可以使69种HPV亚型得到扩增,包括黏膜型HPV、皮肤型 HPV以及疣状表皮发育不良相关性 HPV;PCR反应体系的敏感度在10-2～10-3拷贝之间。41例非生殖器部位Bowen病的组织标本中,5例HPV DNA阳性,其中高危黏膜型3例（2例HPV16,1例HPV33）,皮肤型2例（HPV27和HPV76各1例）。结论 改良的巢式PCR方法具有较高的敏感性、特异性,部分非生殖器部位Ｂowen病发病与黏膜高危型ＨＰＶ感染相关。     

尖锐湿疣皮损中人乳头瘤病毒基因分型研究

目的 采用反向杂交研究尖锐湿疣皮损中人乳头瘤病毒(HPV)的感染状况。方法 提取尖锐湿疣新鲜标本的HPVDNA,采用PGMY09/11引物系统进行聚合酶链反应(PCR)。PCR产物在标记有37种HPV型特异性探针的尼龙膜条带上进行HPVDNA杂交分型。所有DNA模板采用HPV6和11型特异性引物进行PCR检测验证。数据经SPSS11.0统计软件分析。结果 杂交结果显示201例标本HPVDNA均为阳性,共发现31种HPV基因型,其主要的HPV基因型名称及所占比例分别如下:HPV11(53.7%,108/201)、HPV6(43.8%,88/201)、HPV16(6.5%,13/201)、HPV52(6.0%,12/201)、HPV33和HPVcp6108(均为5.5%,11/201)、HPV42(5.0%,10/201)等。60.2%(121/201)的标本由单一型HPV感染,39.8%的标本由混合型HPV感染。HPV6和11型特异性引物PCR结果显示HPV6和11的阳性率分别为45.8%和56.2%,与杂交结果比较,一致性分别为98.5%和96.5%,资值分别为0.97和0.93,P值均<0.001。结论 至少有31种HPV基因型与尖锐湿疣相关。HPV11阳性率最高,HPV68、40、54、67、73、82、35、64和83在尖锐湿疣中少见,HPVcp6108在尖锐湿疣中首次发现,且阳性率较高(与HPV33并列第5位)。HPV26、69、70、71、72和IS39可能与尖锐湿疣不相关。尖锐湿疣中单一型和混合型HPV阳性率分别为60.2%(121/201)和39.8%(80/201)。     

Lack of detection of human papillomavirus DNA in male urine samples.

OBJECTIVES--To evaluate polymerase chain reaction (PCR) methodology for the detection of urethral human papillomavirus (HPV) infection by examining urinary sediment from males. SETTING--Department of Genitourinary Medicine, Leeds General Infirmary. SUBJECTS--73 male patients attending for treatment of sexually transmitted diseases, including 14 patients with genital warts which did not involve the urethral meatus. METHODS--Urinary sediment was tested for HPV DNA and human beta globin gene DNA by PCR methodology. A consensus primer set capable of detecting a wide range of HPV types was used. PCR product was analysed by gel electrophoresis and ethidium bromide staining. RESULTS--HPV DNA was not detected in any of the specimens. Human beta globin gene DNA was identified in 40 of the 73 specimens (55%). CONCLUSIONS--Screening urinary sediment for HPV DNA by PCR methodology with analysis of PCR product by gel electrophoresis and ethidium bromide staining is probably unhelpful for studying the prevalence of urethral HPV infection in men.     

Detection of human papillomavirus in cutaneous extragenital Bowen's disease in immunocompetent patients

INTRODUCTION: A specific link between human papillomavirus (HPV) types 16, 18, 31, and 33 and genital carcinomas and between HPV type 5 and cutaneous extragenital carcinomas in patients with epidermodysplasia verruciformis and renal transplant has been previously found. The aim of this prospective study was to detect HPV in cases of cutaneous extragenital Bowen's disease (BD) from non-immunosuppressed patients. PATIENTS AND METHODS: Twelve cases of cutaneous extragenital BD or Bowen's carcinoma (BC), seen in the period 1994-1996 and confirmed by histologic examination, were included in the study. Tissue sections were studied by in situ hybridization with a mixture of HPV DNA probes and specific HPV DNA probes. In addition, study on fresh materiel from 1995 included: Southern blot hybridization with various usual HPV probes (6, 11, 16, 18, 31, 33, 35, 39, 42), polymerase chain reaction (PCR) with hybridization using consensus HPV probes and probes specific for HPV types 6, 11, 16, 18 and 33. In positive samples with conventional PCR, in situ PCR with probes specific for HPV types 6/11 and 16 was performed on tissue sections. RESULTS: In situ hybridization was negative in all the cases. Southern blot hybridization was negative in our 9 studied cases. Three cases studied by consensus PCR were positive. PCR with specific HPV probes revealed positivity on two of these cases: HPV 6 in one, and HPV 16 in another. In situ PCR was positive with a mixed 6/11 HPV probe in the third positive consensus PCR case. DISCUSSION: Our study revealed the presence of HPV in 3 out of 12 cases of cutaneous extragenital BD and BC. HPV type 16, found in BC of skull, was the most usually found type in the literature. HPV types 6/11, detected in 2 cases, were rarely found in cutaneous extragenital BD and BC and these results are in favor of the oncogenic effect of these virus types. In our study, in situ hybridization and Southern blot hybridization were negative in all the cases; HPV was only found in 3 cases by conventional PCR and in 1 case by in situ PCR. The low range of detection of HPV in cutaneous extragenital BD may be due to the used methods, to difficulties related to sampling and/or to a low number of copies of the HPV genoma.     

光线性角化病皮损中β型人乳头瘤病毒检测及分型

【摘要】 目的 分析光线性角化病（AK）皮损中β型人乳头瘤病毒（HPV）感染情况及型别分布。方法 收集39例AK患者皮损以及40例健康对照皮肤,应用巢式PCR进行β-HPV和α-HPV检测。筛选出β-HPV感染阳性标本,设计12对特异性（HPV5、8、15、17、19、20、21、23、36、38、49和80型）引物,应用普通PCR技术对其分型进行分析。结果 39例AK患者皮损组织β-HPV检出率为84.6%（33/39）,健康对照组为30.0%（12/40）,两组差异有统计学意义（χ2 = 6.76,P < 0.05）。β-HPV分型：病例组以HPV38型检出率最高（36%,12/33）,其次为HPV36。健康对照组此12种β-HPV感染率均较低。病例组HPV混合感染10例,健康对照组均未发现混合感染。病例组不同年龄、性别、职业、病程组间β-HPV阳性率差异均无统计学意义（χ2 = 0.53,0.94,0.81,0.73,均P > 0.05）。病例组α-HPV阳性率为12.8%,对照组为7.5%,差异无统计学意义（χ2 = 0.91,P > 0.05）。结论 AK 患者皮损β-HPV感染率远高于健康对照组,以HPV38型感染最为多见。     

Detection of genital human papillomavirus (HPV) DNA by PCR and other conventional hybridisation techniques in male partners of women with abnormal Papanicolaou smears.

OBJECTIVE--To study the prevalence of human papillomavirus (HPV) infection, using several different hybridisation techniques, in men whose female sexual partners had cervical HPV and/or cervical intraepithelial neoplasia (CIN). METHODS--The male genital area was examined colposcopically and areas suspicious of HPV changes were biopsied. Each biopsy was subjected to histological examination and HPV DNA analysis by conventional DNA analysis such as Southern, reverse and dot blot as well as with polymerase chain reaction (PCR). RESULTS--Colposcopic examination of men showed 133 to be normal whilst 82 (38%) had clinical or subclinical lesions. Of 55 colposcopically directed biopsies from the male lesions taken, detection of HPV DNA by hybridisation with conventional techniques and by PCR showed HPV DNA in 29 (53%) and 47 (85%) of biopsies respectively. Overall HPV types 6/11 were the predominant types. In 18 (33%) biopsies positive by PCR, multiple types were found. CONCLUSION--HPV DNA was present in the majority of biopsy specimens taken, with HPV 6/11 being the predominant type. Among methods for HPV DNA detection, PCR was the most sensitive and useful technique.     

人乳头瘤病毒与皮肤Bowen病的相关性

目的 探讨人乳头瘤病毒(HPV)与皮肤Bowen病发病的相关性.方法 41例皮肤Bowen病患者皮损以及48例健康对照皮肤,采用多对引物,应用巢式PCR进行HPV DNA检测,同时应用半定量PCR进行病毒定量,对HPV DNA阳性标本进一步采用原位杂交方法分析组织内病毒的分布状况.结果 41例皮肤Bowen病患者皮损HPV DNA阳性检出率为12%(5例),其中黏膜型3例(2例HPV16,1例HPV33),病毒定量相当于101～103拷贝,原位杂交显示在多数肿瘤细胞核中有广泛阳性表达,而肿瘤邻近的正常组织无信号表达;皮肤型2例(HPV27和HPV76各1例),原位杂交均无阳性表达.此外,对照组中有1例检出HPV DNA,属于疣状表皮发育不良相关型HPV23,检出率为2.1%,与皮肤Bowen病皮损中HPV检出率比较,差异无统计学意义.2例患者皮肤型病毒定量较低,与正常对照组中检出的1例HPV23相似,均相当于10-2～10-3拷贝.结论 某些皮肤Bowen病的发病与黏膜型HPV密切相关.     

CO2激光碳化尖锐湿疣残存HPVDNA活性的实验研究

对20例尖锐湿疣（CA）患者，局部皮损经CO2激光治疗后，碳末DNA进行PCR扩增，并与正常角质形成细胞（KC）培养，观察KC增殖情况，观察其是否具有活性。结果发现：碳末DNAPCR扩增，40％阳性，碳末DNA对KC有明显抑制作用。从而推断碳末DNA仍有活性，可能是引起该病复发的原因之一。     

Frequency and spectrum of HPV types detected in cutaneous squamous-cell carcinomas depend on the HPV detection system: a comparison of four PCR assays

BACKGROUND: The association of human papillomavirus (HPV) with cutaneous squamous-cell carcinomas (SCCs) has been described recently, but the frequency and spectrum of HPV types identified differed substantially in distinct studies. OBJECTIVE: Comparison of different PCR assays with respect to sensitivity and range of HPV types detected. METHOD: Cutaneous SCC were analyzed for HPV DNA using both consensus PCR assays with degenerate primers and PCR assays with nondegenerate primers derived from HPV types 5 and 8. RESULTS: HPV DNA was found in 50% of SCC specimens using degenerate primers. The rate of HPV-DNA-positive specimens increased to 69% when PCR assays with nondegenerate primers were applied in addition. The spectrum of HPV types detected with each of the PCR assays differed considerably. CONCLUSIONS: The frequency and spectrum of HPV types detected in cutaneous SCC strongly depends on the HPV detection system used and urges the need for standardization of HPV detection and typing in skin lesions in order to characterize HPV types predominating in distinct tumors.     

Identification of human papillomavirus in keratoacanthomas

BACKGROUND: Keratoacanthomas are benign, clinically distinct skin tumors that may infiltrate and show cellular atypia. A viral etiology has been suggested, and the aim was to search for human papillomavirus (HPV) in keratoacanthomas. METHODS: From 21 immunosuppressed organ transplant recipients and 11 non-immunosuppressed patients, 72 fresh biopsies with diagnosis of keratoacanthomas were analyzed. For detection of cutaneous and genital HPV DNA, single-tube nested "hanging droplet" polymerase chain reaction (PCR) and another PCR (GP5+ and 6+) were used, respectively. RESULTS: Among 21 immunosuppressed patients, 71% (15/21) harbored HPV DNA at least in one sample. Of the keratoacanthoma lesions, 55% (33/60) were HPV DNA positive. Fourteen samples from eight immunosuppressed patients contained HPV types 5, 9, 10, 14, 19, 20, 21, 38, 49, 80, putative HPV types as HPVvs20-4, HPVvs75, and HPVvs92 and FA16.1, FA23.2, FA37, FA75, and FA81. Among 11 non-immunosuppressed patients, 36% (4/11) harbored HPV DNA at least in one sample, and 33% (4/12) of their keratoacanthomas were HPV DNA positive. In total, HPV DNA was detected in 51% (37/72) of the keratoacanthomas. CONCLUSIONS: By the use of PCR, cutaneous HPV DNA was detected in 51% (37/72) of the keratoacanthomas. No predominating HPV type or genital HPV type was identified. The role of HPV in keratoacanthomas remains thus elusive.