Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Detection of human papillomavirus (HPV) type 16 and 52b in cervical cancer tissues by Southern blot hybridization and polymerase chain reaction (PCR)

DNA samples from cancer tissues diagnosed histologically as squamous cell carcinoma of the uterine cervix were examined for the presence of human papillomavirus (HPV) genome DNA by Southern blot hybridization. Of 63 specimens, 32 were found to hybridize with HPV DNA probes; 23 specimens (35%) with HPV type 16 (HPV16), one (2%) with HPV type 18 (HPV18), four (7%) with HPV type 52b (HPV52b), and four others (7%) weakly with HPV52b. Specimens negative for HPV DNA with Southern blot hybridization were subjected to polymerase chain reaction (PCR) to determine the presence of HPV DNA under more sensitive conditions. After PCR using one set of primers specific for HPV16 and HPV52b, 7 out of 31 specimens were found to have HPV16 DNA. None was positive for HPV52b DNA. Our results indicate that HPV52b, as well as HPV16 and HPV18, is associated with squamous cell carcinoma of uterine cervix, and more sensitive determination of HPV infection can be made by amplification of the viral genome by PCR.     

Prevalence of selective inhibition of HPV-16 DNA amplification in cervicovaginal lavages

HPV-16 viral load has been assessed with real-time PCR assays by measuring HPV-16 DNA and a human gene in genital samples. HPV-16 viral load measurements are thus based on the inference that inhibitors contained in samples will equally impede amplification of DNA sequences from HPV-16 and human DNA. We have previously shown that sample lysates can inhibit amplification of HPV-16 but not beta-globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative beta-globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV-16 DNA and beta-globin in real-time PCR assays. Of 150 lysates screened with both ICs, 12 (8%) contained inhibitors. Inhibition of amplification of both ICs was demonstrated in four of these specimens. In eight lysates, amplification of HPV-16 IC only was impeded. Six (50%) of these 12 lysates tested positive for HPV-16 DNA despite the presence of PCR inhibitors. The HPV-16 viral load increased significantly after dilution of 11 of 12 lysates, demonstrating the presence of inhibitors in the undiluted lysate. Nine (90%) of 10 samples with inhibitors that were tested after dilution did not demonstrate inhibitory activity. The use of internal controls in real-time PCR is clearly essential to determine HPV viral loads since the effect of inhibitors on primer-driven genomic amplification is variable.     

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay.

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.     

Intra-laboratory reproducibility of human papillomavirus identification in cervical specimens by a polymerase chain reaction-based assay.

BACKGROUND: polymerase chain reaction (PCR)-based assays for human papillomavirus (HPV) sequences are in wide use in clinical and epidemiological studies. The reproducibility of these assays is not extensively studied. OBJECTIVES: to estimate the intra-laboratory reproducibility of generic and type-specific HPV diagnoses by the MY09/MY11/HMB01 consensus L1 primer-based PCR assay. Study design: systematically collected specimens (n=207) were masked and retested. RESULTS: when specimens negative in both initial and repeat assays were excluded from analysis, the diagnostic reproducibility was 98. 6% for beta-globin, 90.7% for generic HPV (any HPV type), and 76.9% for type-specific HPVs. The reproducibility of type-specific diagnosis increased with increase in signal strength in the hybridization reaction of the initial assay. When a specimen contained five or more HPV types in the initial assay, it was rare to identify all of the HPV types in the repeat assay. CONCLUSIONS: the degree of reproducibility of the PCR diagnosis should be taken into account in the interpretation of HPV data in clinical and epidemiological studies.     

Increased detection rate of human papillomavirus in cervical scrapes by the polymerase chain reaction as compared to modified FISH and southern-blot analysis

Cervical scrapes from 80 women with a positive cytology result were tested for the presence of human papillomavirus (HPV) using the polymerase chain reaction (PCR) and compared to the results obtained with the modified filter in situ hybridisation (FISH) and the Southern-blot techniques. The sensitivity of the modified FISH and the Southern-blot was similar, and HPV was detected in 46% of the patients. The sensitivity of the PCR appeared to be higher, and HPV was detected in 70% of the patients. HPV-DNA could be detected in 46 of the 68 patients with mild dysplasia, in 6 of the 8 patients with severe dysplasia, and in all 4 patients with carcinoma in situ. In 18 patients (21%) more than one HPV type could be detected by the PCR. The control group consisted of 100 women involved in a triennial checkup programme, who had normal smear results and no history of cervical lesions. HPV was detected in 5% of the women by the PCR. The PCR technique detected HPV in a high proportion of the cervical scrapes from women with a positive cytology result. These results give further evidence for an important role of HPV in the pathogenesis of cervical cancer.     

Comparison between vaginal tampon and cervicovaginal lavage specimen collection for detection of human papillomavirus DNA by the polymerase chain reaction

The aim of the study was to compare the accuracy of self-administered vaginal tampon (VT) specimens for the detection of human papillomaviruses (HPVs) with that of cervicovaginal lavage specimens (CVL). Two hundred seventy-four paired VT and CVL specimens were collected prospectively from women at risk of sexually transmitted diseases. Specimens were treated and amplified with the polymerase chain reaction (PCR). Each woman served as her own control. One hundred and forty-four of 272 (52.9%) CVLs and 159 of 271 (58.7%) VTs contained HPV DNA sequences (correlation of 88%). The sensitivity and specificity of vaginal tampons reached 93.9% (138/147) and 80.5% (99/123), respectively. HPV typing results were concordant for 99 negative paired samples and 114 paired samples positive for the same type(s) (correlation of 78.9%). It is concluded that these sampling methods collect cells from different areas of the genital epithelium, highlighting the importance of further assessment of the comparative predictive value of HPV detection in each sample. J Med Virol 51: 42–47, 1997. © 1997 Wiley-Liss, Inc.     

Prevalence of HPV cervical infection in a family planning clinic determined by polymerase chain reaction and dot blot hybridisation

The overall prevalence of human papillomavirus (HPV) cervical infection in 131 women attending a family planning clinic was 7% (HPV 6/11, 16, 18, 31) by dot blot hybridisation, 53% (HPV 11, 16, 31) by polymerase chain reaction (PCR), and 56% by the two methods combined. HPV 16 and 18 were the commonest types (4% each) by dot blot, HPV 16 (39%) by PCR. Fifteen percent of subjects had mildly abnormal cervical cytology (grades 1A, 2A, or 3). There was no significant correlation between cytological abnormality and HPV positivity, or between cytological or HPV status and other postulated risk factors for cervical neoplasia. It is concluded that PCR is considerably more sensitive than dot blot DNA hybridisation in detecting HPV cervical infection in such a "low risk" setting, where HPV copy number may be low. Firm conclusions cannot be drawn from our results regarding a causal role for HPV or other factors in the development of cervical neoplasia.     

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: A method suitable for semiautomation

Screening for high-risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow-up. Thirty-four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high-grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR-enzyme-linked immunosorbent assay (ELISA)-based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR-ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR-ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia. © 1996 Wiley-Liss, Inc.     

Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction.

Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggests that some risk factors such as particular sexual habits, that are inversely related to age, must exist.     

HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Analysis of prostate tissue DNA for the presence of human papillomavirus by polymerase chain reaction,cloning, and automated sequencing

We have analysed the DNA from 24 prostate tissue biopsies, spanning a range of Gleason grading from benign to grade 5 and mixed randomly with cervical cancer samples of known human papillomavirus (HPV) status, for the prevalence of HPV DNA, in a double-blind study to ensure complete objectivity. Polymerase chain reactions (PCR) were performed using general E1 open reading frame primers for HPV under low stringency conditions, in addition to reactions containing primers specific for HPV16, E2, and E6 open reading frames under higher, more stringent PCR conditions. The presence of cellular DNA was verified by the use of primers for hypoxanthine guanine phosphoribosyl transferase. DNA bands were not detected in the prostate biopsies using the HPV16-specific primers under high-stringency PCR conditions, however a predominant band in the 400 bp region was observed in 15 of the prostate biopsies using the general primers and the low annealing temperature of 40°C. This fragment was excised and cloned into the pT7 blue vector and the sequence of the insert determined. Although the cloned sequences initiated and terminated with the two authentic PCR primers, they did not contain a significant HPV-related open reading frame. Our results indicate that HPV type 16 and closely related types, as detected by the general primer pair, are unlikely initiators of prostate carcinogenesis within our population. J. Med. Virol. 52:8–13, 1997. © 1997 Wiley-Liss, Inc.     

2400例宫颈细胞脱落标本HPV筛查及荧光PCR基因分型和定量

目的建立快速、灵敏的HPV筛查方法及探讨HPV基因型与宫颈癌之间的关系.方法用细胞涂片法和传统PCR对2400例年龄在18～65岁的厦门郊区妇女进行筛查,对PCR阳性标本进一步进行荧光PCR基因分型和定量.结果 2400例宫颈脱落细胞普查中有宫颈原位癌4例、CIN(癌前病变)Ⅰ～Ⅲ级7例;PCR定性筛查这些标本中有84例阳性,阳性率为3.5%.对这84例阳性标本进行荧光基因分型和定量,发现13例HPV-16、18型阳性,其中HPV-16型阳性8例(占9.52%),HPV-18型阳性5例(5.95%),并且HPV-DNA拷贝数在3.6×103到5.6×107之间.在HPV-16、18型阳性的患者中宫颈原位癌3例(占75%)、CINⅠ-Ⅲ级5例(占71%).结论 HPV-16、18型与宫颈癌之间有高度相关性.     

Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction.

A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100%, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%.     

Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay

Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays. Cells were inoculated on the CAM of 9-day-old chick embryos and the membrane at the opposite side of the egg was recovered after 48 h incubation. To measure intravasation ability, we calculated the amount of human DNA in each CAM sample by real-time PCR of Alu sequences and SYBR Green I fluorescence detection. This analysis showed a detection limit of 1 human cell per 10⁵ total cells, and we were able to distinguish between tumor cells of distinct invasive capacity. This assay has several advantages over current methods to measure intravasation ability, including the elimination of post-PCR analysis, sensitivity and easy scale-up of sample numbers. This revised version was published online in July 2006 with corrections to the Cover Date.     

聚合酶链反应快速检测胃活组织标本中的幽门螺杆菌

幽门螺杆菌是一些胃、十二指肠疾病的主要原因之一。将聚合酶应用于临床诊断，检测胃活组织标本中的幽门螺杆菌，取得了满意的结果。ＰＣＲ可检出少至０．１ｐｇＨｐＤＮＡ，相当于１００个细菌细胞。一次ＰＣＲ检测可在５小时内完成。     

Consensus polymerase chain reaction and enzyme-linked immunosorbent assay for human papillomavirus detection and typing in cervical specimens.

Human papillomavirus (HPV) infection is common in cervical intraepithelial neoplasia (CIN). This study investigates HPV detection and typing assay based on polymerase chain reaction amplification of L1 open reading frame with general primers GP5/GP6, followed by enzyme-linked immunosorbent assay detection with type-specific DNA probes. To determine the sensitivity of this assay, formalin-fixed CaSki cells were used as reference cell lines. Fifty copies of viral DNA diluted in DNA from 100,000 noninfected cells could be detected. This assay was also investigated for HPV detection and typing of 67 cervical specimens diagnosed with with CIN III or carcinoma in situ (CIS) and their adjacent squamous epithelium. The CIN III lesions were infected in approximately 80% of the samples, 81% in the neighboring CIN II, and 68% in CIN I. The HPV infection was even detectable in 54% of nondysplastic epithelium located near a CIN III lesion.     

Detection of adenovirus DNA in peripheral blood mononuclear cells by polymerase chain reaction assay

Adenovirus can establish persistent infections which may reactivate and cause disease in immunocompromised hosts. Lymphocytes have been postulated to serve as a site of adenoviral persistence based upon the ability to isolate adenovirus from tonsils and to detect adenovirus DNA by Southern blot hybridization in peripheral blood mononuclear cells (PBMC). To test this hypothesis, a more sensitive and specific polymerase chain reaction (PCR) assay was developed to detect adenovirus DNA. Two sets of nested primers were designed to conserved sequences in the adenovirus E1A and hexon genes. The E1A and hexon primers amplified DNA from representative adenoviral serotypes in all six adenoviral groups (A-F). Both primers detected a single copy of the adenovirus type 2 genome but were less sensitive for the group B type 35. None of 33 PBMC specimens from healthy adults and only one of 40 pediatric samples was positive (at a low level) for adenovirus DNA by nested PCR assay. In comparison, PBMC from two children with fatal adenoviral infection were both strongly positive for adenovirus DNA. It is concluded that, in contrast to a previous study, PBMC are not a common site of persistent group C adenoviral infection. In addition, assay of PBMC by the adenovirus-specific PCR may help detect early invasive disease and warrants further evaluation. J. Med. Virol. 51:182–188, 1997. © 1997 Wiley-Liss. Inc.     

子宫颈组织中人乳头瘤病毒16型E6基因片段的定量PCR检测

目的探讨子宫颈组织中人乳头瘤病毒(HPV)16型E6基因的含量与疾病严重程度的关系.方法用定量聚合酶链反应(PCR)检测20例慢性宫颈炎,6例宫颈非典型增生,18例宫颈癌组织中HPV16E6基因的拷贝数.结果HPV16E6基因在慢性宫颈炎,宫颈非典型增生及宫颈癌组织中的平均拷贝数(拷贝/μgDNA)分别为6.16×104,5.33×106和6.45×106.统计学处理表明,宫颈非典型增生及宫颈癌组织中E6基因的拷贝数显著高于慢性宫颈炎(P<0.01),宫颈癌组织中E6基因的拷贝数高于宫颈非典型增生组织,但差异无显著性(P>0.05).结论HPV16E6基因的拷贝数与宫颈疾病程度呈正相关,定量检测HPV16E6基因可作为监测宫颈癌高危人群的一种方法.