用聚合酶链反应检测食管癌组织中人乳头瘤病毒DNA

应用聚合酶链反应（ＰＣＲ）技术对汕头市区６８例食管癌的石蜡包埋标本进行人乳头瘤病毒（ＨＰＶ）ＤＮＡ序列检测，结果显示，ＨＰＶＤＮＡ总阳性率为６６．１８％（４５／６８），检出型别主要为ＨＰＶ６、１１、１６，检出率分别为２７．９４％、３６．７６％和２７．９４％，经统计学处理三型间无显著性差异；ＨＰＶ－１８及未定型别各占８．８２％。值得注意的是ＨＰＶ感染中多重感染占阳性病例的５３．３３％（２４／４５）。初步结果表明，汕头市食管癌高发区有较高的ＨＰＶ感染率，此与食管癌的发生，可能有密切关系。     

应用通用引物聚合酶链反应技术检测结肠癌组织中…

应用人乳头瘤病毒通用引物介导的聚合酶反应技术检测了１５例结肠癌石蜡包埋病理组织切片中ＨＰＶＤＮＡ，其中１０例呈阳性扩增。１２例正常结肠组织经上述ＰＣＲ检测均呈阴性反应。阳性扩增产物经核酸斑点杂交进行ＨＰＶ型别分析，ＨＰＶ１６型占４例，１８例１例，１６／１８型５例，未检出其他ＨＰＶ型别。表明ＨＰＶ可能对结肠癌的发生具有病原相关性。     

应用聚合酶链反应检测食管癌组织中人乳头瘤病毒

目的 探讨人乳头瘤病毒(human papillomavirus,HPV)与我国河南地区食管癌发生的相关性.方法 应用HPV L1通用引物GP5+/6+、HPV16E6和HPV18E6型特异性引物多聚酶链反应(Polymerase chain reaction,PCR),检测林州市食管癌组织中HPV存在状况.结果 31例食管癌组织中,29例检测到HPV阳性,阳性率为93.5%;其中19例检测到HPV16 E6基因,阳性率为61.3%,8例为HPV18 E6基因阳性,阳性率为25.8%,5例HPV16E6和18E6基因均阳性,为混合感染.HPV16和18型阳性率为71.0%.结论 我国河南省林州市食管癌组织中有HPV存在,并且HPV感染可能是食管癌发生的重要病因.     

用通用引物聚合酶链反应对宫颈刮片中人乳头瘤病毒感染的快速筛检

用通用引物聚合酶链反应（ＧＰ－ＰＣＲ）可同时对人乳头瘤病毒（ＨＰＶ）６、１１、１６、１８、３１、３３等型进行检测。该方法成功地用于临床宫颈刮片样品广谱ＨＰＶ感染的筛检。结果，宫颈癌组ＨＰＶ检出率（８５．５％）明显高于非癌组（１３．９％）（Ｐ＜０．０１）；对宫颈癌放疗前后ＨＰＶ阳性率比较，二者亦有明显差异（８５．５％，４３．７％，Ｐ＜０．０１）。证明ＧＰ－ＰＣＲ是一种在大规模人群中快速筛检多型ＨＰＶ感染的有效方法。     

不同引物介导的聚合酶链反应检测人乳头瘤病毒DNA

应用３对人乳头瘤病毒（ＨＰＶ）引物对１０７例各种宫颈标本进行了聚合酶链反应（ＰＣＲ）扩增。结果显示，共同引物（ＧＰ）扩增，有５８．８％（３０／５１）宫颈鳞癌，１００％（１４／１４）尖锐湿疣、１３．６％（３／２２）宫颈炎和１０％（２／２０）正常宫颈出现ＨＰＶ阳性。型特异性引物ＳＰ１６／ＳＰ１８扩增，有３７．２％（１９／５１）宫颈鳞癌２５．７％（５／１４）尖锐湿疣和５％（１／２０）正常宫颈出现ＨＰＶ１６型阳性，５．８％（３／５１）宫颈鳞癌为ＨＰＶ１８型阳性。进一步用ＳＰ１６ｂ引物扩增，没有１例ＨＰＶ１６ｂ亚型被发现。说明宜颈磷癌和尖锐湿疣与ＨＰＶ感染有关，结合应用共同引物和型特异性引物可作为ＨＰＶ检测与分型方法。     

用通用引物聚合酶链反应对宫颈刮片中人乳头瘤病…

用通用引物聚合酶链反应可同时对人乳头瘤病毒６、１１、１６、１８、３１、３３等型进行检测。该方法成功地用于临床宫颈刮片样品广谱ＨＰＶ感染的筛检。结果，宫颈癌组ＨＰＶ检出率，明显高于非癌组；对宫颈癌放疗前后ＨＰＶ阳性率比较，二者亦有明显差异。     

用聚合酶链反应检测结肠癌人乳头状瘤病毒基因

目的　应用聚合酶链反应,检测人乳头状瘤病毒(Humanpapillomavirus,HPV)基因与结肠癌及癌区周边组织的相关性。方法　将结肠镜检获取的72例活检标本进行病理检测,其中结肠癌46例,非癌26例(结肠癌周边组织标本10例),标本用蜡块包埋与固定液两种方法固定,用聚合酶链反应(PCR)特定DNA片段体外扩增法。结果　结、直肠癌人乳头状瘤毒基因检测阳性率434%,结、直肠癌周边组织阳性率10%,非癌组织阳性率为0%。结论　癌区组织基因(HPVs)检测率较高,与非癌对照组相比,差异有显著性(P<0.05),癌组织中HPVs主要以HPV1633型和HPV18型为主,统计学分析表明结、直肠癌的发生、发展与HPV感染有一定的相关性,尤以HPV16型关系更为密切。     

用聚合酶链反应（FCR）技术检测尖锐湿疣组织中人乳头瘤病毒DNA

用聚合酶链反应(polymerase chain reaclion,PCR)技术对40例女性下生殖道尖锐湿疣组织中人乳头瘤病毒(human papilloma virus，HPV)6B／11DNA进行了检测，其中33例阳性(82．5%)。结果表明，PCR技术是当前检测尖锐湿疣中HPV感染快速、特异、灵敏的检测方法。     

人喉癌组织中人乳头瘤病毒DNA的检测

目的为探讨喉癌与人乳头瘤病毒（ＨＰＶ）感染的关系和ＨＰＶ在喉癌中基因组型的分布与表达。方法应用聚合酶链反应技术（ＰＣＲ）制备非放射性探针标记物－地高辛标记ＨＰＶ共有引物探针,对１４６例喉不同病变的新鲜组织标本（喉癌６８例,喉其它病变４８例,正常喉组织３０例）,进行ＨＰＶ６,１１,１６,１８,３１,３３,３５,４２,５８共９型ＨＰＶＤＮＡ感染的检测;阳性者用多重引物ＰＣＲ方法分型。结果喉癌ＨＰＶ感染阳性率４５．６％（３１／６８）,喉癌颈转移淋巴结组织阳性率２０．０％（３／１５）,喉癌前病变阳性率１１．８％（２／１７）,声带息肉阳性率６．３％（１／１６）,１５例癌旁及１５例癌周正常喉组织均为ＨＰＶＤＮＡ阴性。ＨＰＶＤＮＡ型别分布在喉癌中以ＨＰＶ１６、１８型为主,喉良性病变中以ＨＰＶ６、１１型为主。结论喉癌发生与ＨＰＶ感染有关。     

应用原位PCR技术检测阴茎癌组织中人乳头瘤病毒DNA

目的：研究人乳头瘤病毒（ＨＰＶ）与阴茎癌的关系。方法：应用原位ＰＣＲ技术对４６例阴茎癌组织中的ＨＰＶ１６和ＨＰＶ１８ＤＮＡ进行检测。结果：３３例阴茎癌中检测到了ＨＰＶＤＮＡ（７１．７％），其中２９例ＨＰＶ１６ＤＮＡ阳性（６３．０％），６例ＨＰＶ１８ＤＮＡ阳性（１３．０％），２例ＨＰＶ１６和ＨＰＶ１８ＤＮＡ均阳性，４例ＨＰＶ１８ＤＮＡ阳性而ＨＰＶ１６阴性；２例淋巴结转移癌，３例癌旁不典型增生组织和１例癌旁增生组织ＨＰＶ１６ＤＮＡ阳性。结论：阴茎癌与ＨＰＶ１６、ＨＰＶ１８感染有密切关系，原位ＰＣＲ技术是一项敏感性高、特异性强的技术。     

Detection of Human Papiliomavirus DNA in lnvasive Cervical Cancers by the Polymerase Chain Reaction and Its Clinical Significance

In order to detect human papillomavirus (HPV) DNA in invasive cervical cancers, three different polymerase chain reactions to amplify different subgenomic fragments of HPV DNA were carried out on DNA extracted from 93 formalin fixed and paraffin-embedded tumor tissues. This study detected HPV DNA in 54 cases (58.1%), which broke down to HPV 16 in 39 (41.9%) cases, HPV 18 in six (6.4%), HPV 52 in three, HPV 33 in one and unclassified HPV type in the remainder. Histopathologically, squamous cell carcinomas frequently contained HPV 16, whereas, HPV 18 was present in adenocarcinoma, adenos-quamous cell carcinoma and small cell carcinoma of the cervix. Clinicopathological study revealed that HPV 16 and 18 DNA found were more frequently than other HPV subtypes in premenopausal patients. Moreover, HPV 18 DNA positive cancers had a relatively high recurrence rate. These results indicate that cervical cancers might be clinically influenced by the difference in subtypes of the infecting HPV. Acta Pathol Jpn 42: 876–883, 1992.     

使用PCR—SSP方法检测HLA—DR抗原

目的　采用顺序特异引物聚合酶链反应 (PCR -SSP)建立人类白细胞抗原DR位点的DNA分型方法 .方法　合成 2 9个特异性引物和 1对阳性对照引物 ,组成 2 0个PCR反应用于DR位点 ,建立一步法PCR -SSP .结果　所有样本PCR -SSP基因分型获得成功 ,分型结果经标准DNA ,限制性核酸内切酶分析证实符合 ,特异性和重复性 10 0 % .结论　PCR -SSP检测HLA -DR的方法具有快速、准确、特异性高等优点 ,适合临床应用 .     

利用双扩增聚合酶链反应检测非甲非乙型肝炎患者血清中丙型肝炎病毒RNA

利用丙型肝炎病毒（ＨＣＶ）５’－端序列合成两对引物，建立了灵敏、特异的ＨＣＶＲＮＡ双扩增聚合酶链反应检测方法。用此方法及第二代Ａｂｂｏｔｔ酶联抗－ＨＣＶ检测试剂盒，检测了４４例非甲非乙型肝炎患者血清及１０名抗－ＨＣＶ阴性健康人。在４４例患者中，４１例（９３％）ＨＣＶＲＮＡ阳性，３６例（８２％）抗－ＨＣＶ阳性，３３例（７５％）ＨＣＶＲＮＡ、抗－ＨＣＶ全部阳性。３例ＨＣＶＲＮＡ阴性，但抗－ＨＣＶ阳性，另外，有８例抗－ＨＣＶ阴性，ＨＣＶＲＮＡ阳性。１０名健康人ＨＣＶＲＮＡ均为阴性。结果表明，大部分（９２％）抗－ＨＣＶ阳性患者带有ＨＣＶ，但为了检测所有病毒血症患者，抗－ＨＣＶ检测是不够的，利用双扩增ＰＣＲ方法检测ＨＣＶＲＮＡ对于抗－ＨＣＶ阴性患者的诊断是非常有用的。     

用热启动聚合酶链反应检测血清中乙型肝炎病毒DNA技术的建立和应用

建立了热启动聚合酶链反应（ＰＣＲ）检测乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）的技术。ＰＣＲ所有反应成分被２次加样。先加Ａ液（含ｄＮＴＰｓ、一对引物和ＭｇＣｌ＿２）与一粒石蜡珠。７０℃加热后冷却至室温，使蜡珠先融化后凝固形成蜡盖封住Ａ液，然后在向其上加入Ｂ液（含耐热性ＤＮＡ聚合酶、ＨＢＶＤＮＡ模板和ＫＣｌ）并开始循环扩增。当反应管内第一次变性温度升至６０℃以上时，中隔蜡层融化，蜡上浮形成防蒸发屏障，Ａ、Ｂ两液则由于热分子运动而混匀，从而保证引物与靶基因在较高温度下严格退火以减少错导非特异性扩增和引物聚体形成。提高了ＰＣＲ的特异性和灵敏性，应用这种技术对７６例肝炎血清进行了检测，结果ＨＢＶＤＮＡ检出率为６８％，其中ＨＢｓＡｇ（＋）、ＨＢｅＡｇ（＋）、抗－ＨＢｃ（＋）血清检出率为１００％；ＨＢｓＡｇ（＋）、抗－ＨＢｅ（＋）、抗－ＨＢｃ（＋）血清检出率为７０％；ＨＢｓＡｇ（＋）、抗－ＨＢＣ（＋）血清检出率为８９％；抗ＨＢＣ（＋）血清检出率为对％；标记全阴性血清检出率为３３％。     

诺如病毒遗传组I型TaqMan—MGB探针实时荧光RT-PCR的研究

目的建立诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏的检测方法，为疾病预防控制提供可靠的依据。方法根据GenBank诺如病毒遗传组I型代表株保守序列设计特异引物对和TaqMan-MGB探针，建立一步法实时荧光RT-PCR快速检测反应体系，优化反应条件，评价反应体系的灵敏度、特异性、重复性．并与常规RT—PCR比较。结果诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR检测时限短。仅1h就出结果。与轮状病毒、腺病毒、星状病毒、甲肝病毒、诺如病毒遗传组Ⅱ型无交叉反应，最低检出下限为100拷贝／反应，比常规RT—PCR灵敏100倍，5份浓度不同的诺如病毒遗传组I型标本重复检测5次。平均Ct值变异系数范围为0．39％-1．02％。结论诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT—PCR快速、特异、灵敏、重复性好，可应用于突发公共卫生应急检测和诺如病毒遗传组I型监测，提高快速检测能力。     

Multiplex Polymerase Chain Reaction for the Detection of High-risk-Human Papillomavirus Types in Formalin-fixed Paraffin-embedded Cervical Tissues

Detecting high-risk-human papillomavirus (HPV) types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR) for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.     

Detection of Clarithromycin-Resistant Helicobacter pylori by Polymerase Chain Reaction Using Residual Samples from Rapid Urease Test

Background: Approximately 50% of the world population is infected with Helicobacter pylori, which corresponds to a high infection rate. Furthermore, the incidence of antibiotic-resistant H. pylori has increased with the recent rise in use of antibiotics for H. pylori elimination, suggesting growing treatment failures. Aim: The study was aimed to assess the use of residual samples from rapid urease test (RUT) for biomolecular testing as an effective and accurate method to detect antibiotic-resistant H. pylori. Settings and Design: This study was a retrospective study performed using data obtained from medical records of previously isolated H. pylori strains. Materials and Methods: RUT was conducted for 5440 biopsy samples from individuals who underwent health examination in South Korea. Subsequently, 469 RUT residual samples were randomly selected and subjected to polymerase chain reaction (PCR) to detect antibiotic-resistant H. pylori. Statistical Analysis Used: The Chi-square test was used to analyse categorical data. P < 0.05 was considered statistically significant. Results: The results showed a concordance between the results of PCR and conventional RUT in 450 of 469 samples, suggesting that the H. pylori PCR test is a time- and cost-effective detection method. Conclusions: This study demonstrated that PCR test can aid physicians to prescribe the appropriate antibiotics at the time of diagnosis, thus preventing the reduction in H. pylori eradication due to antibiotic resistance, averting progression to serious diseases and increasing the treatment success rate.     

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Targeting 47kDa Gene for the Diagnosis of Scrub Typhus

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October–February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.     

荧光定量聚合酶链反应检测慢性乙肝患者HBV DNA的意义

目的探讨荧光定量聚合酶链反应(FQ-PCR)检测慢性乙肝患者血清乙型肝炎病毒(HBV)脱氧核糖核苷酸(DNA)的临床意义。方法回顾性分析248例慢性乙肝患者的资料,均采用FQ-PCR技术检测血清HBV DNA,检测乙肝病毒标志物(HBV-M)并对比不同HBV-M患者血清HBV DNA水平;对比不同病情患者血清HBV DNA水平;对比不同HBV DNA表达患者外周血T淋巴细胞亚群水平及异常率,分析患者血清HBV DNA水平与外周血T淋巴细胞亚群水平的关系。结果不同HBV-M患者血清HBV DNA水平对比:HBsAg+HBeAg+HBcAb>HBsAg+HBeAg>HBsAg+HBsAb+HBcAb>HBsAg+HBeAb>HBsAb+HBeAb+HBcAb>HBcAb/HBsAb+HBeAb/HBeAb+HBcAb,除HBcAb、HBsAb+HBeAb、HBeAb+HBcAb血清HBV DNA水平差异无统计学意义(P>0.05),其余每2样本比较差异均有统计学意义(P<0.05);不同病情患者血清HBV DNA水平对比:重度病情患者>中度病情患者>轻度病情患者(P<0.05);不同HBV DNA表达患者CD3+、CD4+、CD4+/CD8+对比,HBV DNA阴性患者>低拷贝患者>高拷贝患者(P<0.05),CD3+、CD4+、CD4+/CD8+异常率对比,HBV DNA阴性患者<低拷贝患者<高拷贝患者(P<0.01);本组患者血清HBV DNA水平与外周血CD3+、CD4+、CD4+/CD8+均呈负相关(r=-0.789、-0.812、-0.706,P=0.012、0.007、0.001)。结论在慢性乙肝患者中FQ-PCR检测血清HBV DNA水平与HBV-M、病情和外周血T淋巴细胞亚群水平均有密切关系。