双色荧光定量PCR快速分型、定量高危型人乳头状瘤病毒

目的针对高危型人乳头状瘤病毒（humanpapillomavims，HPV）亚型的多样性以及感染病毒载量的高低，旨在建立高危型HPV的定量与分型的快速检测方法，为HPV筛查与治疗提供依据。方法利用2种荧光染料分别标记HPV-16、HPV-33／52／58／67和HPV-18／45、HPV-31探针，同时以B球蛋白基因作为内对照，对120份疑为HPV感染的宫颈脱落细胞样本进行I{I＇V分型与定量，定量范围为5×10^1-5×10^7拷贝/ml，以HC-2杂交捕获法作为“金标准”，评价该方法的特异性与灵敏度。结果荧光定量PCR阳性检出率为52．5％（631120），其中HPV-16、HPV-18／45、HPV-31和m．V-33，52，58，67阳性率分别为36．51％（23／63）、11．11％（7／63）、12．70％（8／63）、58．73％（37／63）；HPV感染的平均病毒载量为1．08×10^7拷贝/ml。荧光定量PCR的特异性为100％，灵敏度为81．82％。结论双色荧光定量PCR能快速分型、定量高危型HPV，可用于HPV感染的筛查与宫颈病变程度预测以及疗效观察。     

荧光定量聚合酶链反应检测人乳头瘤病毒6/11型

目的 了解女性阴道炎患者人乳头瘤病毒6／11型（HPV6／11）的感染情况。方法 用荧光定量聚合酶链反应（FQ－PCR）技术检测129例门诊患者阴道分泌物中HPV6／11的病毒拷贝数。结果 共检出HPV6／11阳笥者47例,阳性率为36．43％;拷贝数在10^5以上者39例,占阳性者中的82．98％;40岁以上年龄组阳性率高且病毒复制量均在10^5以上。结论 中老年阴道炎患者就诊晚,感染重。FQ－PCR检测HPV敏感、快速、准确,特别是其定量特点对临床很有意义。     

实时荧光定量PCR检测人乳头瘤状病毒基因

目的了解妇女中人乳头瘤状病毒（HPV）的感染率,为有效防治宫颈疾病发生提供依据。方法采用实时荧光定量PCR检测技术对401例本院妇产科门诊就诊疑似HPV感染者取宫颈分泌物,进行HPV6、11型和HPV16、18型检测。结果总阳性率36.66%,其中HPV6、11型阳性率43.02%,HPV16、18型阳性率24.26%,同时做HPV6、11型和HPV16、18型检测,阳性率14.71%。并且半数妇女年龄集中在25-45岁之间。结论女性感染HPV非常普遍,应给予高度重视。     

宫颈癌与人乳头状瘤病毒16/18型的关系探讨

目的　应用荧光定量聚合酶链反应 (FQ -PCR)检测宫颈癌患者人乳头状瘤病毒 (HPV) 16 / 18型感染率 ,探讨HPV 16 / 18型与宫颈癌的关系。方法　应用荧光探针标记引物的荧光定量聚合酶链反应对 88例宫颈癌患者的宫颈分泌物进行了HPV 16 / 18型检测。结果　 88例宫颈癌患者宫颈分泌物FQ -PCR阳性率为 78% ,阳性样品定量对数平均值 (ml-1)为 5 .33× 10 6,定量测值范围 (ml-1)为 1.2 0× 10 3 ～ 2 .4 1× 10 7。对照组 85例全部阴性。结论人乳头状瘤病毒 (HPV) 16 / 18型感染与宫颈癌的发生发展关系密切。     

荧光定量PCR法检测母乳巨细胞病毒感染和母婴传播

目的了解母乳人巨细胞病毒（HCMV）感染状况和母婴传播情况.方法应用荧光定量PCR（FQ-PCR）法检测390份母乳中HCMV-DNA含量,其中236份母乳配对与患儿血液或尿液中HCMV-DNA含量作一定分析.结果可疑或确诊HCMV感染患儿母亲母乳HCMV-DNA阳性率达71.28%,HCMV-DNA阳性母乳喂养的婴儿,其血或尿HCMV-DNA阳性率明显高于HCMV-DNA阴性母乳喂养的婴儿.结论HCMV感染母乳是婴儿获得性感染的主要途径.     

荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

目的：建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法：利用Bac-to-Bac载体系统在E.coli菌株DH10 Bac中构建重组杆状病毒穿梭质粒（Bacmid）和在昆虫细胞中构建含人IL-18基因的重组杆状病毒,纯化的重组Bacmid作为PCR检测的标准模板,由昆虫细胞中收获的病毒母液用于空斑测定和病毒DNA提取.以10倍梯度稀释的重组Bacmid作为标准模板,进行荧光定量PCR扩增IL18基因片段并绘制标准曲线,然后以提取的重组杆状病毒DNA作为模板,采用同样体系进行实时PCR反应检测.同时,以琼脂糖空斑法测定病毒母液的滴度.结果：成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101～108拷贝,病毒母液的DNA拷贝浓度（vg/ml）值约为空斑检测的滴度pfu/ml值的10倍.结论：荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒,并在较大的线性范围内检测重组杆状病毒滴度,较之空斑法更准确地反映了重组杆状病毒的实际数量.     

实时荧光定量PCR法与杂交捕获法在高危型HPV检测中的比较

大量研究已经证明,人乳头瘤病毒感染(HPV),特别是高危型人乳头瘤病毒感染,在妇女宫颈疾病的发生和发展中起着重要的作用。HPV感染是宫颈癌及宫颈上皮内瘤变(CIN)的主要病因,高危型HPV感染是宫颈癌及其癌前病变的必要条件。据报道[1],宫颈癌患者HPV阳性率可达到99.7%,HPV感染使宫颈癌的相对危险性增加200多倍,是可     

实时荧光定量PCR法筛查唐氏综合征的实验研究

目的建立一种无创性、高特异性的快速筛查唐氏综合征（Down’s syndrome，DS）的检测方法。方法采用实时荧光定量PCR技术对正常人和DS患者外周血样本的有核细胞进行检测，根据△Ct值的差异建立检测方法。结果正常组和唐氏组△Ct值有显著性差异（P〈0．001），初步建立了实时荧光定量PCR筛查唐氏综合征的快速检测方法。结论实时荧光定量PCR具有无创性、特异性高且简单、快速等优点，适用于唐氏综合征产前筛查。     

实时荧光定量PCR在检测人ANP基因表达的应用

目的建立心房钠尿肽（ANP）基因mRNA表达水平的实时荧光定量PCR检测方法,并对该方法进行初步评价。方法以基因表达产物为模板,建立实时荧光定量PCR检测方法,对样本中的心房钠尿肽含量进行相对定量,比较不同样本组的基因表达水平。结果所建立的实时荧光定量PCR方法熔解曲线中熔解峰单一。肺癌患者胸腔样本的ANP含量为对照样本的4.68倍,血清样本为对照样本的16.03倍。结论所建立的ANP实时荧光定量PCR检测方法具有较高的特异性。肺癌患者胸腔液和血清中ANP的含量明显增高。     

吕梁地区宫颈病变患者人乳头状瘤病毒基因型的检测分析

目的调查吕梁地区21种人乳头状瘤病毒基因型的检测分析。方法收集568例女性宫颈病变患者宫颈分泌物中的脱落细胞,应用人乳头状瘤病毒导流杂交快速基因分型技术检测21种人乳头状瘤病毒亚型,包括13种高危亚型(16,18,31,33,35,39,45,51,52,56,58,59和68型)、5种低危亚型(6,11,42,43和44型)和3种中国人群常见亚型(53,66和CP8304型);分析21种基因型的流行病学特征。结果人乳头状瘤病毒感染率41.9%,单一感染率58.4%,混合性感染率39.9%。21种基因型中,高危型以16,53型为主,其次是52,58型,低危型以6,11型为主;人乳头状瘤病毒16型的感染率居首位。结论本地区21种人乳头状瘤病毒基因型的检测分析资料对人乳头状瘤病毒疫苗研究、应用及其感染的防治有重要意义。     

人乳头瘤病毒感染与人乳头瘤病毒疫苗的研究进展

人乳头瘤病毒(HPV)感染与宫颈癌的发生密切相关,是宫颈癌发生的最主要诱发因素.预防性HPV疫苗是一种预防宫颈癌的新方法,其效果得到了多项临床试验的肯定.治疗性HPV疫苗的研发同样备受关注,目前治疗性疫苗的类型很多,但因其机制较复杂,大多仍处在实验阶段.     

宫颈癌患者HPV感染和调节性T细胞的相关性研究

目的 探讨宫颈部位人乳头瘤病毒（HPV）感染与CD4＋ CD25＋ CD127-调节性T细胞（TReg）及机体免疫水平的关系.方法 采用第二代核酸杂交扩增技术（HC2）检测41例正常对照组、50例宫颈上皮内瘤变（CIN）Ⅰ、42例CIN Ⅱ～Ⅲ和70例感染HPV宫颈癌患者;采用流式细胞术检测外周血CD4＋CD25＋ CD127-TReg、细胞毒性T细胞（CTL）和NK百分率;采用酶联免疫吸附实验（ELISA）检测血清中TGF-β1和INF-γ的含量.结果 ①HPV感染率随着宫颈病变的加重而升高（P =5.75×10^-19）;②宫颈CIN Ⅱ～Ⅲ组与宫颈癌组外周血CD4＋CD25＋CD127-TReg、CTL、NK、TGF-β1、INF-γ与CIN Ⅰ组及正常对照组比较,差异均 有统计学意义（P=1.03×10^-9）;且在HPV阴阳性组间比较亦有统计学意义（P=2.33×10^-4）;③CTL与NK和Treg呈负相关（P值分别为1.62×10^-6和3.41×10^-5）;INF-γ含量与TReg呈负相关（P=2.11×10^-5）;④HPV与CD4＋ CD25＋ CD127-TReg百分率呈正相关（P =3.02×10^-6）.结论 宫颈HPV感染与TReg密切相关,TReg失调导致的免疫水平下降可能是宫颈癌免疫逃避机制之一.     

Identification of epitopes in Indian human papilloma virus 16 E6: a bioinformatics approach

HPV-16 is reported as the cause of cervical and other related carcinomas. The early expressed protein E6 in cancer cells is found to be the target for immune therapeutic methods. The sequence of HPV-16 E6 (Accession No: ABK32509) from NCBI databank has been taken for this study. Hydrophilicity, flexibility, accessibility, turns, exposed surface, polarity and antigenic propensity scales were used for the B cell epitope prediction. MHC Class I and Class II alleles for the accession were predicted by the MHCPred 2.0 Program. The epitope sequences were also found out. Computer-based prediction program results show, A0203 and DRB0101 lower IC50 than other alleles. The best peptide binding affinity was 21HLCTELQTT30 of A0203 allele. In DRB0101 allele the peptide found was 39YCKQQLLRR48. Different structural features of the protein have also been predicted including glycosylation, kinase C phosphorylation, casein kinase II phosphorylation and N-myristylation sites. These computational prediction programs show four glycosylation, five kinase C phosphorylation, two casein kinase II phosphorylation, zero N-myristylation sites and seven disulphide sites. Development and approval of new vaccines are the keys for control of cancer. Epitopes and other structural features of protein prediction could be the best source of information and can help in molecular and medical studies of viral infection and development of HPV associated cancer drugs.     

Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR

To better understand the prevalence of Gallibacterium anatis in different poultry species, a rapid and accurate method was developed to detect G. anatis using a TaqMan fluorescent quantitative polymerase chain reaction (qPCR). Specific primers and a TaqMan probe were designed based on the reference gtxA gene sequence. The qPCR standard curve showed a good linear relationship, and the method showed good reproducibility, sensitivity, and specificity, indicating its suitability for G. anatis identification and quantitative analysis. A comparison of the detection results in 160 clinical swab samples showed that the detection rate (54.4%) of the qPCR for G. anatis was better than that of two conventional methods: gyrB gene-based qPCR for G. anatis (51.9%) and culture-based identification (34.4%). G. anatis was detected in layer chicken (77.3%), Silkie chicken (72.7%), and duck (27.1%) with relatively high detection rates, whereas dove (8.8%) and quail (3.0%) showed lower detection rates, indicating the different prevalence of G. anatis in different fowl species.     

宫颈脱落细胞标本中人乳头瘤病毒16、18型E6基因的检测

观察宫颈脱落细胞标本是否能替代宫颈组织标检测HPV16、18型的感染。通过聚合酶链反应－限制性片段多态性分析。比较127例宫颈患者的单、双份宫颈脱落细胞和宫颈病变组织中HPV16、18型E6基因的检出率。发现单、双份宫颈脱落细胞标本中HPV16型E6基因的检出率分别为34．64％和40．73％；HPV18型的检出率为17．32和22．04％，存在差异。组织标本中HPV16、18型的检出率分别为41．73％和22．83％，与双份脱落细胞本的检出率无明显，表明双宫颈脱落细胞标本可替代宫颈组织标本检测患者宫颈组织中HPV16、18型感染情况。     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR），并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳，EB染色，UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术，用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct，原始拷贝数在10^5/ml以上者为阳性，用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性，阳性率29.2%；用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性，阳性率29.2%；用定性PCR观察到193例有阳性特异带，阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便，灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点，值得在临床检验中推广应用。     

Predictors of human papilloma virus in patients with keratinization

Given the prevalence of human papilloma virus (HPV) infection, an attempt was made to determine whether certain factors such as keratinization and/or squamous atypia are associated with its development. Review of our gynecologic cytology files from 1989 yielded 1,615 specimens showing parakeratosis and/or hyperkeratosis, without cytologic evidence of HPV. Concomitant diagnoses included no atypia [keratinization only (KO)], inflammatory squamous atypia (ISA), and squamous atypia (SA). Morphologic follow-up including repeat cytology or biopsy was available for 916 cases, 92 (10.0%) of which possessed changes of HPV. For any case with both cytologic and biopsy evidence of HPV, only the biopsy result was tabulated. HPV on follow-up examination was detected in 52 (6.7%) of the 764 cases with KO; in 20 (20.8%) of the 96 cases with keratinization and ISA (KISA); and in 20 (35.7%) of the 56 cases with keratinization and SA (KSA). The definitive diagnosis of HPV was based on previously described features (Gupta, In: Comprehensive Cytopathology, Philadelphia: WB Saunders, 1991:133-140) including nuclear enlargement with nuclear membrane irregularities in combination with sharply demarcated paranuclear cytoplasmic clearing. Affected cells have rounded borders. Binucleated cells are not uncommon. The increasing percentage of HPV from KO to KISA to KSA is not necessarily surprising. However, mathematical analysis revealed statistically significant differences in the development of HPV in each of the 3 groups: KISA vs. KO (P < 0.001), KSA vs. KO (P < 0.001), and KSA vs. KISA (P < 0.05). Therefore, a cytologic diagnosis of keratinization with ISA or especially SA should warrant closer follow-up than that of KO.     

阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究

目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。     

Awareness and uptake of human papilloma virus vaccines among female secondary school students in Benin City,Nigeria

BackgroundThere is no Government endorsed HPV vaccine immunisation program in Nigeria. The Vaccine has been available at the University of Benin Teaching Hospital (UBTH) in Benin City for more than 7 years.ObjectivesThe aim was to evaluate awareness about HPV, the prevalence of HPV immunisation and its associated factors among the study population.MethodsA cross-sectional study using interviewer-administered questionnaires among 215 females attending secondary schools in Benin city, Nigeria. Participants were selected using multi-stage stratified sampling. The primary outcome measure was HPV immunisation of the girls.ResultsThe majority of the participants were between 14 to 18 years (58.6%). Almost all the participants (>97%) had not heard of HPV, HPV Vaccines and Cervical cancer. In addition, 2 (0.9%) persons correctly identified that the virus can be transmitted sexually while only 1 person (0.5%) had received the HPV vaccine. The respondents all agreed that they needed to be enlightened about HPV, HPV vaccines and Cervical cancer. Majority (49.3%) of the girls suggested that this could be done through the mass media (49.3%) or their parents (32.1%).ConclusionHPV immunisation, knowledge of HPV vaccines and Cervical cancer among the study population was very low. We recommend interventions in Schools to increase knowledge about cervical cancer and HPV vaccines.     

聚合酶链反应酶谱分型检测宫颈癌中人乳头瘤病毒 …

目的 探讨人乳头瘤病毒（ＨＰＶ）和单纯疱疹病毒（ＨＳＶ）等对宫颈癌的病因学作用。方法 应用聚合酶链反应（ＰＣＲ）－核酸内切酶分型检测宫颈癌活检组织中ＨＰＶ－ＤＮＡ和ＨＳＶ－ＤＮＡ基因,以正常宫颈组织作对照。结果 在宫颈癌活检细胞中ＨＰＶ－１６,１８型和ＨＳＶ－２型阳性率分别为３８．９％和３４．６％,与正常妇女宫颈组织阳性率均为３．２％比较,差异均有非常显著意义（Ｐ〈０．００１）。结论 ＨＰＶ－１６