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技术在小儿巨细胞病毒感染诊断和治疗中的应用

目的 建立小儿人巨细胞病毒（HCMV）感染的分子诊断方法。方法 采用荧光定量聚合酶链反应（FQ－PCR）法测定45例疑诊HCMV感染的患儿，并与常规PCR及酶联免疫吸附试验（ELISA）比较。对确诊为HCMV肝炎的25例分别在5个时点（治疗前、出院时、3个月、6个月及9个月）监测其外周血白细胞中HCMV DNA拷贝（copies)数。结果 常规PCR、ELISA和FQ－PCR的阳性率分别为60．00％，33．33％和66．67％，灵敏度分别为84．38％，46．88％和93．75％。HCMV肝炎治疗组和对照组的HCMV DNA量在5个时点的差异有非常显著意义（P＜0．001）。以10^3 copies/ml的FQ－PCR值为临界值（≥10^3有症状，＜10^3无症状）可预测活动性HCMV感染的出现。结论 FQ－PCR可作为小儿HCMV感染的有效诊断方法之一，动态检测HCMV DNA含量有助于指导治疗、估计疾病的发展和预后。     

不孕患者中宫颈HPV高危型别感染率及病毒载量的调查

目的调查高危型人乳头瘤病毒（HPV）在不孕患者宫颈细胞中的感染情况,探讨HPV病毒型别及其载量（viral load）对不孕发生的影响。方法对临床130份不孕患者的宫颈脱落细胞标本和在门诊体检的150份对照组的宫颈脱落细胞标本采用多通道实时荧光定量PCR仪进行八种高危HPvDNA分型及定量检测,该八种高危HPV型别为主要高危型：HPV16,18,45,31和次要高危型HPv33,52,58,67。结果不孕组阳性率为25．38％（33／130）,对照组的阳性率为11．33％（17／150）,两组间的阳性率差异有统计学意义。不孕组的33份阳性标本中病毒载量≥10^6为24例,病毒载量〈10^6为9例;对照组的17份阳性标本中,病毒载量≥10^6为4份,病毒载量〈10^6为13份,两组间的病毒载量有显著性差异。结论不孕组高危型HPV感染率比正常人群（对照组）高。对不孕组病毒载量的分析表明：不孕组人群中其病毒载量明显高于正常人群。此外,本研究还为分泌物核酸检测的定量设置了内标（β-球蛋白）,提出可供临床使用的分泌物取样的核酸定量检测方法。     

女性生殖道感染治疗前后解脲支原体荧光定量PCR检测的假阳性分析

目的探讨荧光定量PCR（FQ—PCR）检测解脲支原体的假阳性率，为客观分析其结果提供依据。方法采用FQ—PCR及培养法同时检查了70例无症状妇女及163例生殖道感染患者治疗前后宫颈分泌物中解脲支原体。结果无症人群FQ—PCR检测阳性率（42．9％），明显高于培养阳性率（11．4％）（P＜0．01）；生殖道感染患者FQ—PCR检测阳性率（66．9％），高于培养阳性率（45．4％）（P＜0．05）；治疗10天后FQ—PCR检测阳性率（40％），高于培养阳性率（10．8％）（P＜0．01）。结论FQ—PCR检测女性生殖道解脲支原体有一定的假阳性，应用时应客观分析其结果。     

FQ-PCR检测161例女性HPV16、HPV18感染状况报告

目的为了解健康体检妇女和阴道炎患者人乳头状瘤病毒（HPV）16型和18型的感染状况。方法采用荧光定量聚合酶链反应技术，对99例健康体检妇女和62例阴道炎患者进行了HPV16、HPV18检测。结果在上述99例健康体检妇女中检测出、HPV18阳性标本1例，阳性率为1．01％，未检测到HPV16阳性标本；在62例阴道炎患者中检测出HPV16阳性标本6例，HPV18阳性标本1例，阳性率分别为9.68％和1．61％。结论在阴道炎患者中HPV16有较高的感染率。     

先天性HCMV感染新生儿尿液中病毒载量与疾病严重度的关系探讨

目的探讨新生儿尿液中人巨细胞病毒（HCMV）病毒载量与先天性感染的关系及与HCMV所致疾病严重程度的关系。方法采集98例经PCR方法确诊的有症状及无症状的先天性HCMV感染新生儿尿液标本,用实时荧光定量PCR法（FQ—PCR）检测尿液中巨细胞病毒载量。结果98例先天性HCMV感染的新生儿中85例在出生后有临床症状（86％）。无症状感染和有症状感染的新生儿尿液中平均HCMV病毒载量分别是1．4×10^5拷贝／ml和3．1×10^6拷贝／ml,P〈0．01,差异有统计学意义。结论先天性HCMV感染的新生儿中无症状感染者尿液中病毒载量显著低于有症状感染者,提示病毒载量与疾病严重程度相关。     

尖锐湿疣患者HPV亚型及IL2检测

目的：检测尖锐湿疣（CA）患者人乳头瘤病毒（HPV）亚型及白介素2（IL2）。方法：用PCR检测61例CA患者皮损中的HPV6／11、HPV16／18、HPV－DNA。用双体抗夹心ELISA法检测58例CA患者外周血IL2。结果：HPV6／11阳性40例；HPV16／18阳性12例；HPV－DNA阳性、HPV6／11阴性、HPV16／18阴性9例。HPV16／18阳性组IL2低于HPV6／11阳性组，两组相比差异有显著性（P＜0．05）。结论：HPV16／18感染比HPV6／11感染有更强的抑制IL2产生的作用。推测CA患者的HPV16／18感染比HPV6／11感染更易复发。     

包皮中人乳头瘤病毒基因的检测

了解人乳头瘤病毒（HPV）在正常包皮中的感染及感染型别，来分析HPV与阴茎癌的发生关系。采用PCR技术对48例包皮环切患者的包皮组织进行HPV感染相关性研究及分型。20／48例HPV阳性，阳性率为41．7％，其中HPV11、16、18型阳性率分别8．3％（4／48）、12．8％（6／48）、22．9％（11／48），未见HPV6型。包皮垢或包皮炎患者也有HPV11、16、18型感染。正常包皮中有HPVDNA存在且多为高危型，并伴有包皮垢或既往包皮炎等局部理化刺激因子，为HPV感染与阴茎癌的发生有密切关系提供了佐证。     

荧光定量PCR检测沙眼衣原体及临床应用

目的：用荧光定量聚合酶链反应（FQ－PCR）检测沙眼衣原体的含量,探讨FQ－PCR在衣原体性尿道炎诊断中的价值。方法：应用荧光探针标记引物的荧光定量聚合酶链反应对172例疑为沙眼衣原体感染患者标本进行检测,并与常规PCR（电泳－EB染色）进行比较。结果：FQ－PCR阳性率为19．8％,常规PCR法为20．9％,两法符合率为63．9％。女性宫颈分泌物标本FQ－PCR阳性为29．2％,常规PCR法为16．7％;男性分泌物FQ－PCR阳性率为16．9％,常规PCR法为13．8％。FQ－PCR特异性较常规PCR法高。结论;FQ－PCR在扩增中实时在线检测,并选择理想的标准曲线做出较精确的临值分析,具有较高的特异性和敏感性,对病原体的诊断有一定的临床意义。     

荧光定量聚合酶链反应检测血清中HBV-DNA

目的 :研究荧光定量聚合酶链反应 (FQ PCR)检测HBV DNA对辨别病毒复制的临床意义。方法 :采用FQ PCR和ELISA方法 ,检测 79例患者血清HBV DNA拷贝数和免疫学血清指标。结果 :2 6例HBsAg、HBeAg、抗 HBc均阳性的标本 ,HBV DNA也全部阳性 ,平均拷贝数为 2 .7× 10 5/mL。 8例HBsAg、HBeAg阳性的标本 ,HBV DNA也全部阳性 ,平均拷贝数为5 .2× 10 4/mL。 2 1例HBsAg、抗 HBe、抗 HBc均阳性的标本HBV DNA阳性率为 6 6 .7% ,平均拷贝数为 3.1× 10 4/mL。 9例HBsAg阳性的标本HBV DNA阳性率为 11%。平均拷贝数为1.4× 10 3 /mL。结论 :荧光定量聚合酶链反应检测HBV DNA可以更加准确地判断HBV的感染和复制情况。     

Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.     

Comparative analysis of human papillomavirus detection by polymerase chain reaction and Virapap/Viratype kits

Cervical samples from 270 women referred by area physicians were analyzed for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by the polymerase chain reaction (PCR) followed by Southern hybridization. Samples from 154 patients were concurrently analyzed by a commercial filter hybridization technique (Virapap and Viratype Kits, Life Technologies, Bethesda Research Labs, Gaithersburg, MD). The sensitivity of the Southern blot procedure combined with PCR was significantly higher than that of the Virapap and Viratype methods. HPV was detected in 67% of women who had positive results for dysplasia by PCR and in 47% by the Virapap method. HPV types 16/18 were found more commonly than types 6/11 in every diagnostic category. More than one HPV type was detected in 12% of HPV-positive patients. The prevalence of HPV in cytologically negative or indefinite patients as measured by PCR was 22% and 40%; in contrast, by the Virapap method, these values were 7% and 10%. These results demonstrate that PCR combined with Southern hybridization provides a higher level of sensitivity than methods that use hybridization without amplification of HPV DNA and also show that the prevalence of HPV is highest in cytologically positive smears.     

Prevalence of human papillomaviruses in urine samples of male patients infected with HIV‐1 in Sao Paulo,Brazil

Human papillomavirus is a DNA virus that includes 118 genotypes. HPV16 is responsible for 80% of cervical cancer in women. Men are important reservoirs and major transmitters of HPV to their partners. The aim of this study was to detect HPV DNA and to determine the prevalence of HPV types 6, 11, 16, and 18 in urine samples of men infected with HIV‐1. This study included 223 patients infected with HIV‐1 from the Center of Reference on HIV/AIDS (CRT‐SP) and an outpatient clinic of HIV. Urine samples were collected and after DNA extraction real‐time PCR was performed for detection of HPV DNA. Positive samples were then tested by conventional PCR using type‐specific primers for the four HPV types. A total of 223 men infected with HIV‐1 were tested, 81% of whom were on HAART. Four (5.8%) were positive for HPV6, 18 (26.1%) were positive for HPV11, 22 (31.9%) were positive for HPV16 and five (7.2%) were positive for HPV18 by conventional PCR. Twenty (29%) patients had other HPV types and five patients (1.5%) had multiple types. The mean T CD4+cells count was 517 and 441 cells/mm³ (P = 0.30), in HPV negative and positive men, respectively. The HIV viral load was higher in the HPV negative group than for in the men with HPV (P = 0.0002). A 30.9% prevalence of HPV was found in asymptomatic urine samples of men infected with HIV‐1. This study suggests that urine may be a useful specimen for HPV screening. J. Med. Virol. 81:2007–2011, 2009. © 2009 Wiley‐Liss, Inc.     

Absence of human papillomavirus DNA in nongenital seborrheic keratosis

Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology, human papillomavirus (HPV) has been suggested as a possible causative agent. Viral involvement, however, has not been confirmed yet despite extensive research. The aim of this study was to evaluate the presence of HPV 6/11, 31, 33 DNA in nongenital SK. We analyzed 40 biopsy specimens taken from patients with nongenital SK using in situ polymerase chain reaction (PCR) and PCR with tissue extracts. The SK specimens (n=40), analyzed by in situ PCR, were negative for all HPV probes tested (types 6/11, 31, 33). Control slides (condyloma acuminatum, n=3) were positive for type 6/11, 31, and 33 HPV probes tested. Melasma samples (n=4), the negative controls, were consistently negative. No HPV DNA band was detected by PCR with the tissue extracts from paraffin-embedded SK samples, while condyloma acuminatum, the positive controls, showed DNA bands of the correct molecular weights. Our results show that HPV type 6/11, 31, and 33 cannot be recognized as causative agents for nongenital SK, which is in contrast to the previous studies. Further studies are required to reveal the presence of other types (more than 90) of HPV DNA.     

Typing of human papillomaviruses in condylomata acuminata from Greece

DNA samples from recurrent condylomata acu-minata biopsies of Greek males and females were examined for the presence of human papil-lomavirus (HPV) DNA using high-stringency Southern blot hybridization analysis. Of the twenty-six biopsies, 25 were positive for the HPV 6/11-related DNA sequences, and when further analyzed with the polymerase chain reaction (PCR) the HPV-negative biopsy was also positive for HPV 6/11 DNA. Nineteen specimens were further characterized based on their Pstl restriction endonuclease hybridization pattern. Twelve biopsies were positive for HPV 6a, one biopsy was positive for HPV 11a, and one biopsy was positive for HPV 6c DNA. Three specimens contained HPV 6/11 related DNA that gave an unusual Pstl pattern, and one specimen appeared to represent a multiple HPV infection containing HPV 6/11- and HPV 31/35/39-related sequences. Finally, one sample contained a mixture of HPV 6a DNA and an HPV 6a-like genome. Biopsies were also taken from adjacent apparently normal tissue, 0.5 cm away from the lesion, in 19 of the patients. Only one of these was found to be positive for HPV 6a DNA by Southern blot analysis. © 1994 Wiley-Liss, Inc.     

乙型肝炎病毒 C 基因启动子基因变异与免疫学标志及DNA含量的临床研究

目的 研究乙型肝炎病毒（HBV）C基因启动子（Basic core promoter,BCP）基因变异与HBV感染者免疫学标志及HBV DNA含量的临床关系。方法 采用聚合酶链（PCR）微板核酸分子杂交及荧光定量PCR检测技术,对246例HBV感染者进行HBV BCP基因变异及HBV DNA含量测定。结果 在HBsAg/HBeAg/HBcAB,HBsAg/HBeAg/HBcAb及HBsAg/HBcAb阳性的HBV感染者中,HBV DNA的阳性率分别为97.5%(48/49),22.9%(25/109),31.1%(14/45);HBV BCP基因变异率分别为59.1%(29/49),11%(12/109),13.3%(6/45);HBV BCP基因变异组DNA含量均≥10^6cps/ml,明显高于非BCP基因变异组。肝硬化病人BCP基因变异明显高于其他组。结论 e抗原免疫学标志的转换仅对部分感染者预示着病毒的免疫清除和静息,单独依靠乙型肝炎免疫学标志并不能确切提示HBV的复制状态、病变程度及愈后。     

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.     

Failure to detect human papillomavirus DNA in malignant epithelial neoplasms of conjunctiva by polymerase chain reaction

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of conjunctival tumors, 44 formalin-fixed, paraffin-embedded specimens of conjunctival tumors (24 patients with papillomas and 20 patients with dysplastic and/or malignant tumors) were screened for HPV infection using 4 different polymerase chain reactions (PCRs). Of the 24 samples of papilloma, 14 (58%) displayed positive results by applying nested PCR using primer sets of HPV consensus L1 region. HPV type 6 or 11 was detected in 9 cases of papilloma by type-specific primer sets, but none of them were positive for HPV type 16 or 18. However, by using the highly sensitive PCR technique, we failed to demonstrate the HPV DNA of HPV types 6, 11, 16, and 18 in any of the 20 malignant epithelial tumors of conjunctiva. We conclude that HPV-6 or HPV-11 is present in a substantial percentage of conjunctival papillomas, which is in accordance with findings of previously reported studies. In contrast, malignant conjunctival carcinomas are not associated with HPV infection; other pathogenic mechanisms, such as UV light, probably are more important in the cause of these malignant lesions.     

High prevalence of human papillomavirus type 58 in patients with cervical pre‐malignant lesions in southern Brazil

Cervical cancer is the second most common type of cancer in women worldwide. Several human papillomavirus (HPV) genotypes, sexual behavior, and socioeconomic profile represent major risk factors for the development of this carcinoma. Cervical invasive cancer is preceded by cellular abnormalities that can be identified by cytological or histological exams. In order to determine the prevalence and genotypes of HPV in women with abnormal cytology or histopathology, cervical cell samples from 256 patients were evaluated for the presence of HPV/DNA by polymerase chain reaction (PCR), followed by virus genotyping by restriction fragment length polymorphism (RFLP). A total of 113 samples (51.2%) were HPV/DNA positive. Viral genotyping showed that the most prevalent genotypes were HPV 16 (34.7%) and 58 (13.8%), followed by HPV 33 (9.72%), 11 (8.33%), 18 (5.55%), 53 (5.55%), and 6 (4.2%). Four samples (5.55%) exhibited multiple infections due to the great similarity of socioeconomic characteristics and sexual behavior of HPV positive women, it was not possible to establish a risk profile for female HPV infection. J. Med. Virol. 81:1270–1275, 2009. © 2009 Wiley‐Liss, Inc.     

Detection of specific types of human papillomavirus in cervical scrapes, anal scrapes, and anogenital biopsies by DNA hybridization

Specific varieties of human papillomavirus (HPV) infecting the anogenital region were detected in clinical samples by use of a filter hybridization technique suitable for rapid screening of cervical and anal scrapes. In this way possibly benign types (HPV6 and HPV11) could be differentiated from types thought to be capable of malignant transformation (HPV 16 and HPV 18). Cervical or anal canal cells were applied directly to nylon filters and fixed by u.v. irradiation before hybridization with mixed viral DNA probes under both low- and high-stringency conditions. In addition, probe for the human Alu-repeated DNA sequence was used to assess the relative amount of total nucleic acids in each sample applied to the filter. HPV DNA was detected in 3 of 19 cervical scrapes from patients with no past or present history of wart virus infection or cervical dysplasia. Within a positive study group totalling 71 patients, HPV (6/11 or 16/18) was detected in cervical scrapes from 24% of 41 patients who did not have visible genital dysplasia, 30% of 27 patients with visible genital dysplasia or cervical intraepithelial neoplasia (CIN) I, and in 1 of 3 patients with past CIN II/III. In addition, HPV6/11 or 16/18 DNA was detected in anal scrapes from 3 of 6 male patients and in 85% of genital biopsies. A notably high proportion (4/6) of vaginal condylomata were positive with both the HPV6/11 and the HPV16/18 mixed viral DNA probes. Of the biopsies prepared for histopathology and positive for HPV DNA, the HPV group-specific antigen could be detected in only 60%.