用光敏生物素标记探针检测宫颈癌、尖锐湿疣及假性湿疣组织中HPV DNA

人乳头瘤病毒(简称HPV)能引起人皮肤及粘膜的多种良恶性肿瘤,特别多见于女性生殖系统如宫颈癌、尖锐湿疣和假性湿疣。但由于HPV尚不能在体外细胞培养中增殖,因此目前对其研究主要用核酸杂交和PCR方法,检测HPV DNA。本文采用光敏生物素标记HPV16、HPV18 DNA探针,分别对宫颈癌组织进行斑点杂交,检测宫颈癌组织中HPV16、18DNA并以正常宫颈组织做对照。以同样方法用HPV6b DNA探针检测尖锐湿疣及假性湿疣组织中HPV6b DNA。结果如下:18例宫颈癌组织中仅有1例与HPV16 DNA探针杂交阳性(5.7%);10例与HPV18杂交刚性(55.6%);4例为HPV16+18混合杂交阳性(22.2%)。12例尖锐湿疣组织中8例与HPV6b DNA杂交阳性(66.7%);12例假性湿疣组织中有6例与HPV6b探针杂交阳性(50%)。10例正常宫颈组织对照均为阴性。     

HPV和HCMV在慢性宫颈炎病人中的分布及相关性研究

本文采用DNA-DNA分子杂交技术对病理组织学确诊的87例慢性宫颈炎患者和25例健康宫颈的宫颈活检组织DNA进行了HPV 6、11、16、18型DNA及HCMV HindⅢE片段检测。结果表明,对照宫颈检出率全部为0,慢性宫颈炎检出率HPV 6为16％,HPVll为12.6％,HPV16为11.5％,HPVl8为5％,总的HPV DNA相关序列为29.0％,HCMV为12.64％。经显著性检验,HPV DNA相关序列检出率在慢性宫颈炎与对照组间具显著性差异,HPV DNA相关序列阳性者患慢性宫颈炎的危险性为HPV DNA相关序列阴性者20.81的倍。本实验结果还表明HCMV阳性患者中72.7％的病人同时有HPV感染,提示HCMV感染与HPV感染有关。     

构建皮肤组织中特异表达HPV16-E6基因的小鼠模型

目的:构建特异在皮肤表达乳头瘤病毒(HPV16)E6基因的真核表达载体,并鉴定其在转基因小鼠体内的表达。方法:通过PCR方法扩增皮肤特异启动子p INV及HPV16-E6,将以上片段通过酶切连接,插入去掉CMV启动子的pc DNA3.1(-)载体,获得dpc DNA3.1(-)-p INV-E6载体;并显微注射制备其转基因小鼠,利用RT-PCR、Western blot及免疫组化技术检测获得的阳性小鼠体内E6的表达水平。结果:dpc DNA3.1(-)-p INV-E6载体测序正确;经鉴定31只实验小鼠中,有2只小鼠携带外源基因,将其与野生型小鼠交配获得的F1代中又有2只阳性小鼠;且在获得阳性小鼠的皮肤组织中RT-PCR检测有E6的转录本,Western blot检测有E6蛋白表达,且免疫组化检测结果显示有E6在皮肤表达且引起皮肤微增生。结论:成功构建了p INV-E6转基因模型小鼠,HPV16-E6基因在小鼠皮肤中特异表达,为进一步研究HPV16-E6在癌症中的作用奠定了基础。     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1 VLP两种抗原在检测宫颈癌抗 16L1或VLP抗体及在宫颈癌血清学诊断意义上的差别 ,应用PCR技术从宫颈癌组织的DNA中扩增出全长15 35bp的HPV16L1基因片段 ,克隆至 pUC18 T载体中 ,进行DNA测序鉴定。然后 ,将HPV16L1基因克隆至pGEX 2T表达载体中 ,并诱导表达HPV16L1融合蛋白 ,分子量为 83kD ,能被HPV16L1单克隆抗体所识别。经GST柱层析法纯化后 ,与重组腺病毒表达的HPV16L1 VLP分别经酶联免疫吸附 (ELISA)法检测 12份宫颈癌患者和 35份献血员血清。 12例宫颈癌血清标本中 ,抗HPV16L1蛋白的抗体阳性率为 7例 (占 5 8.3% ) ;抗HPV16L1 VLP的抗体阳性率为 8例 (占 6 6 .7% )。经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体IgG( )的 7份患者血清 ,利用HPV16L1 VLP试剂盒检测均阳性 ;经大肠杆菌表达的重组抗原检测为HPV16抗体IgG( )的 5份患者血清 ,利用HPV16L1 VLP试剂盒检测有 1份阳性。两者对HPV16抗体的阳性检出率并无显著差异 (P >0 .0 5 )。本实验结果说明HPV16与宫颈癌高度相关 ,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1 VLP重组抗原检测抗体的敏感性并不受影响。利用重组抗原HPV16L1对宫颈癌的抗体进行定性、定量分析有助于该疾病     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1-VLP两种抗原在检测宫颈癌抗16 L1或VLP抗体及在宫颈癌血清学诊断意义上的差别,应用PCR技术从宫颈癌组织的DNA中扩增出全长1535bp的HPV16L1基因片段,克隆至pUC18-T载体中,进行DNA测序鉴定.然后,将HPV16L1基因克隆至pGEX-2T表达载体中,并诱导表达HPV16L1融合蛋白,分子量为83kD,能被HPV16L1单克隆抗体所识别.经GST柱层析法纯化后,与重组腺病毒表达的HPV16L1-VLP分别经酶联免疫吸附(ELISA)法检测12份宫颈癌患者和35份献血员血清.12例宫颈癌血清标本中,抗HPV16L1蛋白的抗体阳性率为7例(占 58.3%);抗HPV16L1-VLP的抗体阳性率为8例(占 66.7%).经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体 IgG(+)的 7份患者血清,利用HPV16L1-VLP试剂盒检测均阳性;经大肠杆菌表达的重组抗原检测为HPV16抗体 IgG(-)的5 份患者血清,利用HPV16L1-VLP试剂盒检测有1份阳性.两者对HPV16抗体的阳性检出率并无显著差异(P>0.05).本实验结果说明HPV16与宫颈癌高度相关,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1-VLP重组抗原检测抗体的敏感性并不受影响.利用重组抗原HPV16 L1对宫颈癌的抗体进行定性、定量分析有助于该疾病的诊断.     

外阴硬化性苔癣组织P53、PCNA表达、DNA含量及与细胞增殖的关系

探讨外阴硬化性苔癣组织中的 P5 3、PCNA表达 ,DNA含量与细胞增殖的关系。免疫组化方法测定 2 0例外阴硬化性苔癣组织和 10例正常外阴皮肤中 P5 3、 PCNA蛋白表达 ;图像分析技术检测两组基底层细胞核形态及 DNA含量。结果显示 ,外阴硬化苔癣组 P5 3阳性表达率为 40 % ,与正常皮肤比较 P<0 .0 5 ,PCNA阳性表达率为 70 % ,与正常皮肤比较 P>0 .0 5 ,阳性表达主要分布于棘层、颗粒层 ;基底细胞核显著变小和 DNA含量降低 (P<0 .0 5 )。结果表明外阴硬化性苔癣组织中存在细胞增殖异常     

广西柳州18～45岁女性人乳头瘤病毒16/18型中和抗体流行率及其与宫颈病变相关性的研究

阐明广西柳州地区自然人群中18~45岁女性人乳头瘤病毒(HPV)16/18型中和抗体和DNA流行情况,并探讨其与子宫颈癌癌前病变的相关性。2013年3月至7月在柳州市招募2 300名18~45岁女性,采集血清以假病毒中和试验(PBNA)法检测HPV16/18型中和抗体,同时采集宫颈脱落细胞进行液基细胞学诊断和HPV DNA检测,对细胞学异常者进行阴道镜检查,并对采集的组织学标本进行病理诊断。采用趋势性χ2检验分析不同年龄段HPV DNA阳性率及HPV中和抗体阳性率的差异,Logistic回归分析筛选宫颈癌前病变的影响因素。广西柳州地区18~45岁女性自然人群中,HPV16DNA或中和抗体阳性364例(15.8%,95%CI:14.4,17.4),HPV18DNA或中和抗体阳性164例(7.1%,95%CI:6.1,8.3)。CIN3在不同年龄组的卡方趋势检验有统计学意义(P=0.005),HPV16和HPV18型DNA阳性是CIN1+(宫颈上皮内瘤变1级及以上)、CIN2+(宫颈上皮内瘤变2级及以上)的主要危险因素,而自然感染产生的中和抗体与癌前病变未发现统计学相关性。HPV16/18型感染是宫颈癌癌前病变的主要危险因素,并未发现自然感染产生的中和抗体与其相关,表明接种疫苗仍是18~45岁女性预防HPV感染及癌前病变的主要方式。     

尖锐湿疣样本中HPV病毒的分子检测

调查男性和女性尖锐湿疣样本中人乳头瘤病毒（HPV）的检出率及病毒类型,为研发相关防治疫苗提供依据,以HPV 外壳蛋白DNA序列为模板设计特异引物,SSP－PCR扩增检测样本中HPV的感染率和病毒类型。收集了北京及邯郸市医院门诊尖锐湿疣样本22例,其中男性13例,女性9例。检测发现所有样本中存在着高浓度的HPV病毒DNA。男性样本中有5例感染HPV6型,6例感染HPV11型,2例为HPV6＋HPV11混合感染。女性样本中有3例感染HPV6型,2例感染HPV11型,4例为 HPV6＋HPV11混合感染。被诊断为宫颈湿疣的4位女性还在其含宫颈粘膜脱落细胞的样本中检出了HPV16、HPV18、HPV33、HPV35、HPV45、HPV54、HPV56或HPV58等高危险型病毒类型。所有检测到的HPV病毒DNA片段均TA克隆并将测定的DNA序列存入了国际基因数据库GenBank（DQ003066－DQ003079）。调查没有在单纯的男女尖锐湿疣组织块中检测到除HPV6和HPV11以外的其他HPV类型。该研究建立了灵敏可靠的HPV分子检测及分型方法,尖锐湿疣中HPV的检出率达100％。 本研究初步结果显示导致男女尖锐湿疣的HPV病毒类型没有显著差异,主要为HPV6及HPV11型。     

宫颈疾患中人乳头瘤病毒和疱疹病毒Ⅱ型DNA的检测

本文应用HPV11,16,18型和HSV-2N/BglⅡ、HSV-2L/HindⅢDNA片段等五个分子探针,通过斑点杂交技术对79例宫颈疾患(包括50例宫颈癌和29例宫颈糜烂)组织DNA进行了检测,结果发现宫颈癌组织HPV16,18和11的阳性率分别为44％,12％和4％,而宫颈糜烂组织中HPV16,18和11的阳性率分别为14％,7％和14％;且3例标本HPV16和HPV18均呈弱杂交反应;在被检的所有宫颈癌组织中各有2例分别与HSV-2N/BglⅡHSV-2L/HindⅢ弱杂交,宫颈糜烂组织无一例阳性。结果提示,HPV在宫颈癌的发生过程中可能起主要作用,HSV-2的作用尚不确定,可能与HPV起协同作用。     

乌鲁木齐地区乳腺癌与人乳头瘤病毒亚型感染的相关性研究

目的：对本地区乳腺癌患者癌组织进行人乳头瘤病毒（HPv）检测,对HPV亚型与乳腺癌的相关性进行研究。方法：选择本地区2010年1月-2013年1月182例乳腺癌患者作为研究组,同时选择30例乳腺良性肿瘤患者作为对照组,均分型基因芯片检测系统对提取的DNA进行分型检测。观察两组患者HPV感染情况及感染亚型。结果：两组患者HPV阳性病例67例,其中研究组阳性66例,阳性率为36．26％,对照组阳性1例,阳性率为3-33％;研究组阳性率明显高于对照组,两组比较差异具有显著性（P〈0．05）。亚型检测结果显示,共检出7种HPV亚型,其中高危型5种,分别为HPV16、HPV18、HPV58、HPV51和HPV56型,低危型两种,分别为HPV6、11两种。研究组高危型61例,低危型5例,对照组1例为HPV6。Ⅰ期、Ⅱ期、Ⅲ期、Ⅳ期乳腺癌患者HPV阳性率分别为16．28％、19．71％、62％、100％,Ⅲ期、Ⅳ期患者的阳性率明显高于Ⅰ期、Ⅱ期,不同分期阳性率比较差异具有显著性（P〈0．05）。结论：乳腺癌患者癌组织中HPV阳性率明显偏高,且绝大多数为高危亚型,而且HPV阳性率与病理分期呈正相关,说明本地区HPV感染与乳腺癌的发生与发展具有一定的相关性,两者的相关性还有待进一步探索。     

DNA sequences of human papillomavirus types 11, 16, and 18 in lesions of the uterine cervix in the west of Scotland.

Punch biopsy specimens of the cervix were examined both histologically and for the presence of human papillomavirus (HPV) DNA sequences. The presence of HPV DNA sequences was sought with the Southern blot technique using radioactively labelled HPV-6, 11, 16, and 18 DNA probes, both together and separately. Twenty six biopsy specimens were examined. Histological examination showed cervical intraepithelial neoplasia grade 2 or 3 in 16 specimens, viral changes (koilocytosis) in four, and inflammation or a normal appearance in three. Eleven specimens were negative for HPV DNA sequences, 10 contained HPV-16 DNA, four contained HPV-18 DNA, and one contained both HPV-18 and HPV-11 DNA. Episomal HPV-16 DNA was detected in one case of cervical intraepithelial neoplasia grade 3 and in five cases of cervical intraepithelial neoplasia grade 2/3 with koilocytosis; and episomal HPV-18 DNA was found in two specimens classed as cervical intraepithelial neoplasia grade 2/3, one of which also contained HPV-11 DNA, and in one specimen that showed viral changes alone. Integrated HPV DNA was found in six specimens (four with HPV-16 DNA and two with HPV-18 DNA), including two cases of chronically inflamed cervix with no histological evidence of viral infection or cervical intraepithelial neoplasia. Detection of viral DNA in early lesions may identify patients at risk of malignant progression. This is the first report of HPV-18 DNA in cervical intraepithelial neoplasia in Scotland.     

Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix.

A previous analysis of 121 female genital tract lesions from the United States and South America had revealed that a large number contained DNA sequences that were weakly homologous to a panel of human papillomavirus (HPV) probes. The DNA sequences of one of these viruses have been molecularly cloned and shown to be a new type of HPV which is called HPV 31. Among the cloned HPV genomes, HPV 31 is most closely related to HPV 16. Although absent from all genital condylomas studied, HPV 31 was present in approximately 20% of the mild and moderate dysplasias and in 6% of the invasive cervical cancers     

Histological and cytological evidence of viral infection and human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and normal tissue in the west of Scotland: evaluation of treatment policy

Biopsy samples from 27 patients referred to a colposcopy clinic in Glasgow for cervical abnormalities were assessed for the relations among colposcopic appearances, cytological and histological diagnosis, expression of papillomavirus antigen, and the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 deoxyribonucleic acid (DNA) sequences. Specimens were from colposcopically abnormal areas of the transformation zone and from colposcopically apparently normal areas of the zone in the same patients (paired matched internal control tissue). All 27 women referred for abnormal smears had colposcopic abnormalities.HPV-16 or 18 DNA sequences were detected in 20 of the 27 colposcopically abnormal biopsy samples and 13 of the 27 paired normal samples. Twelve samples of colposcopically normal tissue contained histological evidence of viral infection but only four of these contained HPV DNA sequences. The other nine samples of colposcopically normal tissue which contained HPV DNA sequences were, however, histologically apparently normal. HPV-6 and 11 were not detected.Integration of the HPV-16 genome into the host chromosome was indicated in both cervical intraepithelial neoplasia and control tissues. In two thirds of the HPV DNA positive samples the histological grade was classed as normal, viral atypia, or cervical intraepithelial neoplasia grade 1. Papillomavirus antigen was detected in only six of the abnormal and three of the normal biopsy samples, and HPV DNA was detected in all of these.The detection of HPV DNA correlates well with a combination of histological and cytological evidence of viral infection (20 of 22 cases in this series). A poor correlation between the site on the cervix of histologically confirmed colposcopic abnormality and the presence of HPV DNA sequences implies that a cofactor other than HPV is required for preneoplastic disease to develop.A separate study in two further sets of biopsy samples examined the state of HPV DNA alone. The sets were (a) 43 samples from cervical intraepithelial neoplasia and nine external controls and (b) 155 samples from cervical intraepithelial neoplasia, cervical cancer, vulval intraepithelial neoplasia, and vulval cancer and external controls. HPV-11 was found in only two (4·7%) of the 43 specimens from cervical intraepithelial neoplasia, whereas HPV-16 was found in 90 (58%) of the other 155 specimens. These results also suggest that HPV subtype is subject to geographical location rather than being an indicator of severity of the lesion or of prognosis.     

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Detection of human papillomavirus (HPV) in genital warts and carcinomas by DNA in situ hybridization in Chinese patients

A series of 51 genital biopsies from normal epithelium, condylomata acuminata, leucoplakia and squamous cell carcinoma from Chinese male and female patients were analysed for the presence of human papillomavirus (HPV) types 6, 11, 16 and 18 by DNA in situ hybridization. All of the nine genital condyloma acuminata were positive for HPV DNA, in which HPV 6 was found in six cases, HPV 11 in two cases and HPV 18 in one case. Twelve out of the 21 cases (57.1% of the total) of cervical squamous cell carcinoma were shown to contain HPV DNA; HPV 16 was found in nine cases, HPV 18 in two cases and HPV 16/18 in one case. Present results support the earlier concept that HPV 6/11 are closely associated with benign genital lesions, and HPV 16/18 are mostly confined to higher grade of intra-epithelial neoplasias and carcinoma.     

Comparative analysis of a liquid-based Pap test and concurrent HPV DNA assay of residual samples. A study of 608 cases

OBJECTIVE: To retrospectively study the HPV DNA assay of residual samples from the ThinPrep Pap Test (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) PreserveCyt (Cytyc) vial as a quality improvement (QI) indicator for management of patients with abnormal cervical cytology. STUDY DESIGN: Six hundred eight residual sample vials of liquid-based Pap-Test specimens were selected for the study based on Pap-test results from October 1998 to March 2001. The specimen vials were forwarded to the reference laboratory (American Medical Laboratories, Chantilly, Virginia, U.S.A.) for HPV DNA assay using the Hybrid Capture System method (Digene Corporation, Gaithersburg, Maryland, U.S.A.). At the time of HPV DNA assay, the residual samples were between 8 days to 10 months old, and each vial contained 4 mL. Of the 608 study cases, 76 were WNL, 115 contained BCC, 172 contained ASCUS, 179 were LSIL and 66 were HSIL. In this study, the 191 WNL and BCC cases were designated as the disease-free control group. The HPV DNA typing results were reported as low-risk, high/intermediate-risk or HPV DNA "not detected" HPV types. The HPV DNA testing results were compared to the Pap-Test diagnoses and statistical analysis performed. RESULTS: The following information reflects the percentage of HPV DNA-positive cases based on the Pap-Test diagnoses: 16.2% in WNL and BCC, 51.1% in ASCUS, 94.4% in LSIL and 98.4% in HSIL. Sensitivity (95.5%), specificity (83.7%), false negative value (4.4%), false positive value (16.2%) and predictive value of a positive (88.3%) and negative (93.5%) Pap-Test were calculated on the basis of HPV DNA testing results for 436 cases that were diagnosed as either SIL or negative (WNL and BCC). ASCUS (172) Pap-Test cases were considered borderline--disease positive and excluded from statistical analysis. CONCLUSION: The HPV DNA assay of residual samples from ThinPrep Pap-Test liquid-based specimens is an objective adjunct to the gynecologic cytology QI protocol and is the gold standard reference test for triaging women with equivocal cytologic diagnoses. The great value of HPV DNA testing is its high sensitivity (95.5%), specificity (83.7%) and negative predictive value (93.5%). HPV DNA testing results can be used as a tool to better determine the need for referrals for colposcopic biopsy, especially for patients with an ASCUS diagnosis. The residual Pap-Test specimens are stable and reproducible for HPV DNA typing. A working flow chart for our gynecologic cytology QI program was produced from the Pap-Test and HPV DNA assay results. This offer presents the added benefit of minimizing the problem of sample variation. The prevalence of HPV infection was 16.2% in this study.     

Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.