原位杂交法检测非甲─非庚型肝炎患者肝组积中新型肝炎病毒DNA

目的证实在非甲-非庚型病毒性肝炎患者肝组织中一种新型肝炎病毒（transfusion－transmittedvirus，TTV）的存在．方法采用地高辛素标记TTVDNA探针以原位杂交技术对51例血清学病毒标记非甲-非戊型、免疫组化检测肝组织中HGVNSS阴性的病毒性肝炎患者石蜡包埋肝组织进行了检测．结果各型病毒性肝炎肝组织中TTV基因的总检出率为27．5％，其中急性轻型肝炎的检出率为30．8％（4／13），急性重型肝炎（1／8，12．5％），亚急性重型肝炎（3／7，42．9％），慢性肝炎（2／6，33．3％），活动性肝硬变（2／9，22．2％），慢性重型肝炎（1／4，25％），原发性肝癌（1／4，25％）TTVDNA表达于肝细胞核或胞浆内，以核型多见．在急性肝炎，TTV阳性细胞弥漫分布于肝小叶内，慢性肝炎于汇管区附近较为密集，而在肝硬变病例，阳性细胞在假小叶内多呈片簇状不规则分布结论在不明原因病毒性肝炎患者血清及肝组织中TTVDNA的检出表明TTV为一种新型的肝炎病毒，TTV为一种嗜肝性病毒。

实时荧光探针定量PCR法在坏死性基质型单纯疱疹病毒性角膜炎病原检测中的应用

背景 单纯疱疹病毒性角膜炎（HSK）是一种常见的感染性眼表疾病,复发率高,重者可致盲,尤其是坏死性基质型HSK.以往对坏死性基质型HSK的诊断主要依靠临床症状和体征,缺乏特异性诊断方法. 目的 检测坏死性基质型HSK角膜上皮组织和泪液中单纯疱疹病毒（HSV） DNA的表达,为坏死性基质型HSK的实验室诊断提供依据. 方法 纳入2012年9月至2013年9月于南京医科大学附属南京医院诊断为坏死性基质型HSK患者30例30眼,裂隙灯显微镜下对临床症状进行炎症评分,予以局部及全身药物治疗8周后,全身用药改为维持量治疗6个月,收集其治疗前和治疗后1、2、4、6和8周的病变区角膜上皮组织和泪液标本,应用实时荧光探针定量PCR（real-time PCR）分别检测角膜上皮组织和泪液标本中HSV的浓度和阳性率,2种组织中HSV阳性率的差异比较采用x2检验,采用多水平混合效应模型比较初诊时角膜上皮组织和泪液中HSK检测结果均为阳性的患眼在各随访时间点HSV的浓度,采用Spearman秩相关法分析患跟治疗前角膜上皮组织中病毒浓度与临床症状体征评分的相关性. 结果 本组30眼治疗前角膜上皮组织和泪液标本中real-time PCR诊断HSV阳性者分别为13例和4眼,阳性率分别为46.4％和13.3％,差异有统计学意义（P=0.006）.治疗后1、2和4周,角膜上皮组织中HSV阳性率均明显低于治疗前,差异均有统计学意义（P=0.001、0.003、0.004）,治疗后6周和8周,均未诊出HSV;而治疗后1周和2周泪液中HSV阳性率与术前比较差异均无统计学意义（P=1.000、0.583）,治疗后4周均未检测出HSV.治疗前30例患者中13例角膜上皮组织和泪液标本HSV阳性者病毒浓度分别为2460（2165 ～ 636500）/ml和0（0～1150）/ml.多水平混合效应模型显示,泪液标本中病毒浓度显著低于角膜上皮组织（P=0.005）,随着治疗的进行,病毒浓度显著下降（P=0.001）,且角膜上皮组织与泪液中下降速率差异有统计学意义（P=0.049）.秩相关分析显示,治疗前角膜上皮组织中病毒浓度与临床症状体征评分值之间呈正相关（rs=0.844,P=0.000）. 结论 Real-timePCR可检测HSK患眼角膜上皮组织中和泪液中HSV的表达,但病变区角膜上皮组织的检测有较高的检出率.治疗前病变区角膜上皮组织较高的病毒浓度者病变更为严重.

慢性乙型肝炎患者外周血单个核细胞内HBV DNA存在的意义

目的探讨外周血单个核细胞（PBMC）内HBVDNA与HBV标志物的关系及在慢性肝病发展过程中的作用．方法应用RCR技术结合斑点杂交技术测定血清HBVDNA及PBMC内HBVDNA血清学标志物采用ELISA法结果在HBsAg＋，HBeAg十组48例中，血清与PBMC内HBVDNA阳性检出率分别为93．8％和83．3％；在HBsAg＋，抗-HBe+组41例中，其阳性检出率分别为48．8％和58．5％.两组间存在显著性差异（P＜0．01）在其他各组中血清及PBMC内HBVDNA检出率均较抵有6例呈单纯PBMC内HBVDNA阳性.在不同类型慢性乙肝患者中，CSH和CMoH组PBMC-HBVDNA检出率分别为78．6％和61．8％，与CMiH组（25．0％）、ASC组（14．3％）比较，存在非常显著性差异（P＜0．01），呈随病情加重而检出率增高的趋势结论PBMC清除HBVDNA较血液缓慢，在病毒持续感染及造成肝细胞损伤过程中可能起一定作用．

HBV-DNA荧光定量检测在原发性肝癌患者中的应用价值

目的 :检测原发性肝癌 (PHC) 14 6例血清中乙型肝炎病毒 (HBV) DNA拷贝数 ,进一步研究 HBV感染与PHC关系。方法 :用 EL ISA法检测 PHC14 6例血清中 HBs Ag、HBs Ab、HBe Ag、HBe Ab、HBc Ab;同时采用荧光定量 PCR法 (FQ- PCR)定量检测其血清中 HBV- DNA含量。结果 :14 6例 PHC中 HBVM总感染率达 90 .4 1%(132 / 14 6 ) ,HBV-DNA总阳性率为 5 6 .85 %(83/ 14 6 ) ,平均 HBV- DNA拷贝数为 10 5.36± 1 .32 / ml。在 HBVM各模式组中 ,小三阳组的病例最多占 4 7.95 %(70 / 14 6 ) ,HBV- DNA阳性率为 71.4 3%,平均 HBV- DNA拷贝数为 10 5.0 5± 1 .2 2 / ml;大三阳组例数较少 (7/14 6 ) ,而 HBV- DNA阳性率最高 (10 0 %) ,平均 HBV- DNA拷贝数亦最高达 10 7.0 2± 1 .4 5/ ml。结论 :本研究发现 HBV感染与PHCS发生关系密切 ,HBe Ab的存在与 PHC之间有相关性 ,而 HBc Ab的持续阳性亦可能是

荧光定量PCR检测HBV-DNA室内质控物的制备及初步应用

目的:制备HBV-DNA荧光定量PCR检测的室内质控物,并对其应用价值进行初步评价。方法:留取单一乙肝模式的混合血清样本,并用正常人血清将收集的血清稀释至所需的浓度,用与待检血清相同的仪器、同一品牌的HBV-DNA定量试剂盒进行检测,确定其靶值,计算x-、s、CV,绘制质控图。结果:自制的室内质控物高低值的变异范围分别为1.10%、1.01%(CV<5%),稳定性好。结论:临床实验室可以用单一乙肝模式的混合血清制备HBV-DNA定量检测的质控物,其制备容易,测定结果稳定,有一定的临床实用价值。

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的:探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性.方法: 采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型,从含有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6,经DNA序列测定法检测其基因变异,进而分析其同源性.结果: 50例宫颈癌组织HPV-DNA的检出率为78%,其中HPV16和HPV18型混合感染18例,单纯HPV16型感染15例.含有HPV16型的标本34例中扩增出HPV16E6 25例.其中178位核苷酸变异较大,变异率为72%,其相应氨基酸均由天冬氨酸变为谷氨酸.结论: HPV16E6 DNA序列发生碱基替换的区域主要在氨基端94～241位,羧基端相对保守,未见变异.广东地区宫颈癌组织中HPV16E6的热点突变为Nt178.

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT_PCR, cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.

阿昔洛韦与IFN序贯抗HBV感染时血清HBVDNA含量变化

目的为探讨HBV感染者单用IFN或阿昔洛韦（Acy）联合IFN序贯治疗时，血中HBVDNA水平变化与疗效关系．方法血清HBsAg，HBeAg，抗HBc：和HBVDNA皆阳性HBV感染者33例．IFN组15例，用重组IFNa-1b300万U，im，1次／d×14，1次／2d×36，共 50支；合用组 18例，用 Acy25 mg／（kg·d），iv计×15，接着 IFNa-1b300万 U，用法同 IFN组，IFN治疗20 d时再用 Acy 15 d，方法同前采用信号引物能量转移定量PCR，测定患者治疗前。中、治疗结束时血清HBVDNA水平并判断疗效．结果完全反应老干扰素治疗前血清HBVDNA含量（拷贝／L）范围 5．03×107～4．58×109；部分反应和无反应者范围 5.68×107～2．37×1015．Acy能降低血中HBVDNA含量，Acy联合IFN序贯治疗组完全反应率（72．2％）明显高于单用IFN组（46．6％）．结论先用Acy降低患者血中HBVDNA含量。

原发性肝癌患者前S1蛋白检测与HBV-DNA的意义

目的了解前S1蛋白与HBVM、HBV-DNA检测的关系及其临床应用价值。方法用ELISA法检测146例PHC患者血清中HBsAg、HBsAb、HBeAg、HBeAb、HBcAb和前S1蛋白;同时采用荧光定量PCR法(FQ-PCR)定量检测其血清中HBV-DNA含量。结果146例PHC患者中HBV-DNA与前S1蛋白的阳性率分别为56.8%(83/146)和45.9%(67/146),两者阳性率差异无显著性;在HBVM各模式组中,小三阳组的病例最多占47.9%(70/146),前S1蛋白阳性率亦最高达67.1%(47/70),超过大三阳组57.1%(4/7)。结论前S1蛋白较HBeAg更敏感的反映了PHC患者体内HBV的复制情况,对HBV标志物及HBV-DNA的检测起着重要的补充作用。

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

抗乙型肝炎病毒核心抗体的临床应用进展

据世界卫生组织报道,全球约2.4亿人为慢性乙型肝炎病毒（hepatitis B virus,HBV）感染者,每年约有65万人死于HBV感染所致肝硬化、肝功能衰竭和肝细胞癌（hepatocellular carcinoma,HCC）[1]。抗乙型肝炎病毒核心抗体（anti-hepatitis B virus core antibody,HBc Ab）是HBV感染人体后的血清免疫学标志之一。

PCR技术检测HBV DNA评价HBsAg阴性消化科住院患者的HBV携带状态

目的为评价消化科HBsAg阴性住院患者是否携带HBV方法使用PCR技术检测了119例HBsAg阴性的消化科住院患者的HBVDNA，并与103例HBsAg阴性传染料住院肝炎患者相比较．结果89．3％的传染科住院患者HBsAg，抗-HBs，抗-HBc，HBeAg，抗-HBe（统称HBVM）阳性，59．2％HBVDNA阳性；52．1％的消化科住院患者HBVM阳性，30．3％HBVDNA阳性．传染料患者两指标都显著高于消化科住院患者（P均＜0．005）HBVM均阴性模式中传染科患者的HBVDNA阳性率显著地大于消化科患者（x^2＝28．92，P＜0.005），两组患者之间HBVDNA阳性率的差异仅存在于HBVM均阴性的HBV感染模式中．结论消化科住院患者中存在NBsAg阴性的HBV携带者，两组HBVM阳性患者实际上来自于同一群体．

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent microscopy.Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after vaccination.The vaccine plasmids disappeared 100 d post-vaccination.Fluorescent microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated tissue.Green fluorescence became weak at 60 days post-vaccination.RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination.These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish.The antigen would therefore potentially initiate a specific immune response.The plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated.The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD.Further studies are required for the development and application of this promising DNA vaccine.

Performance verification and comparison of TianLong automatic hypersensitive hepatitis B virus DNA quantification system with Roche CAP/CTM system

AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the Tian Long automatic hypersensitive HBV DNA quantification system(TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. RESULTS The detection limit of the TL system was 10 IU/m L, and its limit of quantification was 30 IU/m L. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log10 IU/m L, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation(CV) of the same sample was less than 5% for 102-106 IU/m L; and for 30-108 IU/m L, the linear correlation coefficient r2 = 0.99. The TL system detected HBV DNA(A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results(15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed(P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference was-0.49.CONCLUSION The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system in clinical performance.

HPV—DNA在宫颈疾病中的检测价值分析

目的讨论HPV—DNA在宫颈疾病中的监测价值。方法采用二代杂交捕获技术分别检测577例宫颈疾病患者，并借助病理学镜检辅助确诊。结果在上述病例中，HPV—DNA检验出慢性宫颈炎313例（阳性率54．13％）、宫颈上皮内瘤变164例（阳性率78．05％）、HPV亚临床感染92例（阳性率90．22％）、宫颈癌HPV高危型DNA的表达8例（阳性率100％）。结论HPV—DNA高危型检测对早期发现宫颈疾病具有临床意义。

Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma

AIM:Only a minority of patients carrying a defined viral aetiologic agent develop cirrhosis and ultimately hepatocellular carcinoma(HCC),the mechanism underlying the worsening is still undefined.Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and HCC and recently,more Helicobacterspecies(Helicobacter spp.)have been detected in the liver of patients suffering from cholestatic diseases and HCC arising from non-cirrhotic liver.We investigated whether Helicobacterspp.sequences could be detected in the liver of patients with cirrhosis and HCC compared to subjects with metastasis to liver from colon cancer. METHODS:Twenty-three liver samples from patients operated upon for HCC superimposed on hepatitis C virus (HCV)-related cirrhosis and 6 from patients with resected metastases from colorectal cancer,were tested by polymerase chain reaction for presence of genomic 16S rRNA of Helicobacter genus using specific primers.DNA sequencing and cag A gene analysis were also performed. RESULTS:Genornic sequences of Helicobacter spp.were found in 17 of 20(85%)liver samples from patients with HCC and in 2 of 6 samples from patients with liver metastasis. In three samples of the first group the result was uncertain. Hpyloriwas revealed in 16 out of 17 positive samples and Helicobacter pullorum in the other. CONCLUSION:Helicobacter spp.,carcinogenic in mice, were found at a higher frequency in the liver of patients with HCV-related cirrhosis and HCC than those in patients without primary liver disease.