兰金芝,刘扬,徐澍,张金娟,王欢,肖俊,江银辉,陈腾祥
LAN Jinzhi,LIU Yang,XU Shu,ZHANG Jinjuan,WANG Huan,XIAO Jun,JIANG Yinhui,CHEN Tengxiang

• 1:贵州医科大学基础医学院
• 2:贵州医科大学临床医学院
• 3:贵州医科大学贵州省分子生物学重点实验室

摘要(Abstract)：

目的:构建人乳头状瘤病毒(HPV)HPV26和HPV73亚型的单克隆标准品质粒,用于HPV基因芯片制备的质量控制。方法:收集HPV26和HPV73两种亚型感染的临床宫颈刮片脱落细胞样品,提取并PCR扩增样品中HPV的L1区DNA片段,琼脂糖凝胶电泳验证DNA分子量大小;利用基因克隆技术,将L1区DNA片段连接到线性化的p MD18-T质粒载体上,进行琼脂糖凝胶电泳验证质粒连接反应,将构建的HPV质粒转化进入大肠杆菌进行扩增,对质粒进行测序,验证插入DNA序列的正确性;PCR扩增HPV亚型单克隆质粒的L1区特异性DNA片段作为检测标准品,用该标准品与本课题组制备的HPV分型基因芯片进行杂交反应,显色和扫描成像,检测HPV26和HPV73位点的显色情况,从而评估基因芯片HPV26和HPV73探针的特异性和点样质量。结果:从感染HPV患者宫颈刮片脱落细胞成功提取HPV26和HPV73的DNA,并PCR扩增了L1区DNA,琼脂糖凝胶电泳显示扩增的DNA片段符合预期分子大小,将这些DNA片段连接到p MD18-T载体上,电泳结果显示连接后的质粒大小符合预期值,基因测序证实插入的HPV26和HPV73的DNA片段序列正确;PCR扩增了HPV26和HPV73单克隆质粒的L1区特异性DNA片段,电泳显示分子量大小符合预期值;杂交反应显示HPV26和HPV73探针特异性和重现性好,探针的灵敏度为100%,特异性为100%。结论:制备了的HPV26和HPV73的单克隆靶标DNA标准品,可用于HPV基因芯片的质控。
Objective: To construct monoclonal plasmids used as standard sample of HPV26 and HPV73 subtypes of human papillomavirus for quality control of HPV gene chip preparation. Methods:Clinical specimens of exfoliated cells from cervical scrapes of two subtypes of HPV26 and HPV73 infection were collected,and the fragment of HPV L1 region was extracted and amplified by PCR. The molecular weight of DNA was verified by agarose gel electrophoresis. By gene cloning technique,the L1 region DNA fragment was ligated to the linearized p MD18-T plasmid vector,and the agarose gel electrophoresis was performed to verify the reaction of plasmid ligation. The constructed HPV plasmid was transformed into escherichia coli for amplification,and the plasmid was sequenced to verify the correctness of the inserted DNA sequence. The L1 region specific DNA fragment of HPV subtype monoclonal plasmid was amplified by PCR as the detection standard. Hybridization reaction,color development and scanning imaging were performed with the HPV typing gene chip prepared by our team. The specificity and spot quality of HPV26 and HPV73 probes were evaluated by detecting the color development of HPV26 and HPV73 sites. Results: The DNA of HPV26 and HPV73 were extracted successfully from the exfoliated cells of cervical scrapes of infected patients with HPV,and the L1 region DNA was amplified by PCR. Agarose gel electrophoresis showed the expected molecular size of the amplified DNA fragment. These DNA fragments were ligated to p MD18-T vector. The results of electrophoresis showed that the size of the inserted plasmid was in line with the expected value. Gene sequencing confirmed that the sequence of DNA fragments of inserted HPV26 and HPV73 was correct. The L1 region specific DNA fragment of HPV26 and HPV73 monoclonal plasmids was amplified by PCR,and the molecular weight was in line with the expected value by electrophoresis. The hybridization reaction showed that HPV26 and HPV73 probes were specific and reproducible. The sensitivity and specificity of the probes were 100% and 100%,respectively. Conclusion: The monoclonal target DNA standard of HPV26 and HPV73 can be used in the quality control of HPV gene chip.

关键词(KeyWords)： 人乳头状瘤病毒;基因芯片;标准品;质量控制;基因克隆
human papillomavirus;gene chip;standard sample;quality control;gene cloning

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技合作计划项目[黔科合LH字(2016)7348];; 贵阳市人民政府-贵州医科大学联合基金项目[筑科合同(20161001)002号]

作者(Author): 兰金芝,刘扬,徐澍,张金娟,王欢,肖俊,江银辉,陈腾祥
LAN Jinzhi,LIU Yang,XU Shu,ZHANG Jinjuan,WANG Huan,XIAO Jun,JIANG Yinhui,CHEN Tengxiang

DOI: 10.19367/j.cnki.1000-2707.2018.08.006

参考文献(References)：

• [1]STANLEY M.Pathology and epidemiology of HPV infection in females[J].Gynecologic Oncology,2010,117(2Suppl):5-10.
• [2]麦雄燕,韦迪霞,袁飞飞,等.不同基因亚型人乳头瘤病毒感染与宫颈病变的关系[J].中华医院感染学杂志,2018,28(1):121-124.
• [3]李世君,王芳.1 803例女性人乳头瘤病毒基因分型检测及感染情况分析[J].内蒙古医学杂志,2016,48(1):46-48.
• [4]周庆云,王玥元,田芳,等.甘肃地区人乳头瘤病毒基因型与宫颈病变的相关性分析[J].中国生育健康杂志,2011,17(1):5-8.
• [5]魏宝丽,王海平.北京密云地区宫颈液基薄层细胞学检测与人乳头瘤病毒检测对诊断宫颈病变的临床分析[J].中国现代医生,2018,11(2):54-56.
• [6]VILLIERS E M D.Cross-roads in the classification of papillomaviruses[J].Virology,2013,445(1-2):2.
• [7]于春丽.高危型HPV感染与宫颈癌前病变及宫颈癌的相关性研究[J].实用妇科内分泌杂志,2017,4(21):101.
• [8]ZILS K,BIELACK S,WILHELM M,et al.Osteosarcoma of the mobile spine[J].Annals of Oncology,2013,24(8):2190-2195.
• [9]MASROUHA K Z,KHATTAB R,TAWIL A,et al.A preliminary investigation of Beta-h CG expression in patients with osteosarcoma[J].Journal of Bone&Joint Surgery British Volume,2012,94(3):419-424.
• [10]LEE B,SUH D H,KIM K,et al.Utility of human papillomavirus genotyping for triage of patients with atypical squamous cells of undetermined significance by cervical cytology[J].Anticancer Research,2015,35(7):4197-4202.
• [11]CHAN P K,PICCONI M A,CHEUNG T H,et al.Laboratory and clinical aspects of human papillomavirus testing[J].Critical Reviews in Clinical Laboratory Sciences,2012,49(4):117-119.
• [12]VAN D W P,VAN L E,WOLFFS P,et al.Correlation between viral load,multiplicity of infection,and persistence of HPV16 and HPV18 infection in a Dutch cohort of young women[J].Journal of Clinical Virology the Official Publication of the Pan American Society for Clinical Virology,2016,83(6):6-9.
• [13]蒋卫,张艳,王瑛,等.两种人乳头瘤病毒分型检测方法的比较[J].上海交通大学学报:医学版,2008,28(8):962-966.
• [14]LIZARD G,DEMARES-POULET M,ROIGNOT P,et al.In situ hybridization detection of single-copy human papillomavirus on isolated cells,using a catalyzed signal amplification system:GenpointTM[J].Diagnostic Cytopathology,2015,24(2):112-116.
• [15]GONG P,WANG Z,GENG J,et al.Comparative study on detection and typing of human papillomavirus(HPV)infection with microarray using paraffin-embedded specimens from squamous cell carcinoma and cervical precursor lesions[J].Journal of Nanoscience&Nanotechnology,2017,17(2):990-993.
• [16]RAHMAN M,SASAGAWA T,YAMADA R,et al.High prevalence of intermediate-risk human papillomavirus infection in uterine cervices of kenyan women infected with human immunodeficiency virus[J].Journal of Medical Virology,2011,83(11):1988-1996.
• [17]钟泽艳,赵花,钟兴明,等.广东省流动人口育龄妇女HPV感染状况调查[J].中国妇幼保健,2013,28(10):1635-1637.
• [18]周琴,钱文艳,朱艳,等.1 081例患者宫颈HR-HPV感染情况分析[J].现代肿瘤医学,2018,26(6):917-919.
• [19]BZHALAVA D,EKLUND C,DILLNER J.International standardization and classification of human papillomavirus types[J].Virology,2015,476(3):341-344.
• [20]MATSUKURA T,SUGASE M.Pitfalls in the epidemiologic classification of human papillomavirus types associated with cervical cancer using polymerase chain reaction:driver and passenger[J].International Journal of Gynecological Cancer,2010,18(5):1042-1050.

文章评论(Comment)：