生物素标记HBV RNA探针的制备及应用

本文首次采用ＳＰ６５特殊质粒与人类的乙型肝炎病毒ＤＮＡ重组,制备了Ｂｉｏ－ＨＢＶ ＲＮＡ探针,能特异地与ＨＢＶ ＤＮＡ杂交,将该探针与缺口转移方法标记的Ｂｉｏ－ＨＢＶ ＤＮＡ探针进行了比较,结果显示出Ｂｉｏ－ＨＢＶ ＲＮＡ探针比Ｂｉｏ－ＨＢＶ ＤＮＡ探针的敏感性提高１０倍,并分别应用两种探针同时检测７０例乙肝病人血清中ＨＢＶ ＤＮＡ,阳性率各为３１．４２％、２８．５７％（Ｐ＞０．２５）。对Ｂｉｏ－     

乙型肝炎病毒和丙型肝炎病毒的同步PCR扩增

介绍了一种从人血清中同步扩增和检测ＨＢＶ－ＤＮＡ和ＨＣＶ－ＲＮＡ的方法。ＨＣＶ－ＲＮＡ反转录成ｃＤＮＡ,这种ｃＤＮＡ和从ＨＢＶ中抽提出的ＤＮＡ一起,用根据ＨＢＶ、ＨＣＶ保守区序列设计的特异引物进行同步ＰＣＲ扩增,这种方法对于检测ＨＢＶ和ＨＣＶ重复感染很有用处     

HRP-HBVDNA探针在临检应用中的研究

本文介绍了一种简便的检测血清ＨＢＶＤＮＡ的方法。参照Ｒｅｎｚ等人的标记方法,构建了直接酶标ＨＲＰ ＨＢＶＤＮＡ探针。此探针经与固定在硝酸纤维素滤膜上的血清靶ＤＮＡ杂交后,可通过化学发光自显影检测技术观察结果。敏感度可检测０－１ｐｇ靶ＤＮＡ,相当于同位素探针的灵敏度。对６３份ＨＢｓＡｇＨＢｅＡｇ和Ａｎｔｉ ＨＢｃＥＬＩＳＡ阳性血清以及２４份ＨＢｓＡｇＡｎｔｉ ＨＢｃ阳性,ＨｂｅＡｇ阴性血清用ＨＲＰ ＨＢＶＤＮＡ探针进行检测,结果探针ＨＢＶＤＮＡ阳性率分别为１００％（６３）和５８％（１４）;对５０份ＨＢｓＡｇ,ＥＬＩＳＡ阴性和ＡＬＴ正常的血清,探针ＨＢＶＤＮＡ全部阴性。实验结果表明本方法具有很大的推广应用价值。     

HBV－DNA的PCR二步法扩增及其快速检测

ＨＢＶ－ＤＮＡ的ＰＣＲ二步法扩增及其快速检测方勤,吴云涛,蔡宜权（中国科学院武汉病毒研究所,武汉４３００７１）关键词ＨＢＶ－ＤＮＡ,二步温控ＰＣＲ,Ｓｏｕｔｈｅｒｎ杂交,生物素寡聚核苷酸杂交乙型肝炎是危害人类健康的主要疾病之一,其病原常用的检测方法多...     

用套式多聚酶链反应技术监测乙型肝炎病毒的母婴垂直传播

用套式多聚酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ）技术对１６９对ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性孕妇及其新生儿外周血清进行了ＨＢＶ－ＤＮＡ检测。１０３对ＨＢｓＡｇ阳性孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为７２．８％和３３．０％;６６对ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为８６．４％和４３．９％。对５５例ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性产妇产后的初乳进行了ＨＢＶ－ＤＮＡ检测,结果ＨＢＶ－ＤＮＡ阳性率为３６．４％。结果表明ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清ＨＢＶ－ＤＮＡ检出率较ＨＢｓＡｇ单阳性的孕妇及其新生儿要高,其初乳中ＨＢＶ－ＤＮＡ的检出率也高。还对１０５例注射了乙肝疫苗及高价乙肝特异性免疫球蛋白的６月龄婴儿的外周血清进行了ＨＢＶ－ＤＮＡ检测,结果有２３例阳性。     

乙型肝炎病人尿细胞中HBV的实验研究

本文采用间接免疫荧光法（ＩＦ）,ＲＰＨＡ法,ＥＬＩＳＡ法及斑点杂交技术检测１０例无症状ＨＢ－ｓＡｇ携带者及８９例乙肝病人尿细胞中的ＨＢｓＡｇ、ＨＢｅＡｇ及ＨＢＶＤＮＡ,发现尿细胞中有ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ存在。结果提示：乙肝无症状携带者及乙肝病人尿细胞中具有ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ,因此更进一步证实尿液具有传染性。     

乙型肝炎患者HBV复制水平的定量PCR研究

本文采用定量ＰＣＲ方法观察了慢性乙型肝炎患者ＨＢＶ－ＤＮＡ的复制水平。结果表明在ＨＢｅＡｇ阳性患者血清ＨＢＶ－ＤＮＡ检出率和水平显著高于ＨＢｅＡｇ阴性患者（Ｐ＜０．０１）;血清ＨＢＶ－ＤＮＡ水平的高低与ＡＬＴ异常情况未发现有相关性。     

乙型肝炎病人重叠感染丙型肝炎的评价

应用ＥＬＩＳＡ和ＰＣＲ法检测５０２例乙肝病人血清,４０１例ＨＢｓＡｇ阳性血清中,有１１４例（２８．４％）抗－ＨＣＶ和ＨＣＶＲＮＡ双项阳性,２５例（６．２％）ＨＣＶＲＮＡ单项阳性;２１例（５．２％）抗－ＨＣＶ单项阳性。将ＨＢｓＡｇ乙肝病人分成ＨＢＶＤＮＡ,ＨＢｅＡｇ阳性组和ＨＢＶＤＮＡ,ＨＢｅＡｇ阴性组。前者抗－ＨＣＶ阳性率为１１．６％～２０．５％,ＨＣＶＲＮＡ阳性率为１６．２％～２０．５％。后者抗－ＨＣＶ阳性率为２０．２％～５５．６％,ＨＣＶＲＮＡ阳性率为２３％～６０．３％。结果说明长期携带ＨＢＶ者和慢性乙肝病人均可重叠ＨＣＶ感染。ＨＢＶＤＮＡ阳性组抗－ＨＣＶ和ＨＣＶＲＮＡ阳性率明显高于ＨＢＶＤＮＡ阳性组     

乙型肝炎免疫指标全阴的原发性肝细胞癌患者体内HBV—DNA的检测…

本文以聚合酶链式反应（ＰＣＲ）对３５例乙型肝炎免疫指标（ＨＢＶｍ）全阴性的原发性肝细胞癌（ＰＨＣ）患者血清,外周血单核细胞（ＰＢＭＣ）肝活检标本进行乙型肝炎病毒ＨＢＶ－ＤＮＡ检测,其检出率分别为３４．２９％,６０．００％,６８．５７％,说明一部分血清ＨＢＶｍ全阴性的ＰＨＣ患者血清并非无传染性,ＥＬＳＩＡ法具有一定局限性。ＰＢＭＣ内有相当的ＨＢＶ－ＤＮＡ存在,这对于研究ＰＨＣ患者体内ＨＢＶ的存在情况     

单纯疱疹病毒的PCR直接基因分型

本文利用ＰＣＲ技术建立一种对ＨＳＶ直接基因分型的方法。在ＨＳＶ－Ⅰ、Ⅱ两型的ＤＮＡ多聚酶基因上设计了一条两型共同的上游引物（ＨＤＰ－Ｂ）和两条型特异的下游引物（ＨＤＰ－１、ＨＤＰ－２）。三条引物共同组成一个扩增反应体系,在ＨＳＶ－Ⅰ产生５４３ｂｐ条带,ＨＳＶ－Ⅱ产生３７２ｂｐ条带,据此在基因水平上对ＨＳＶ进行分型。５株不同来源的ＨＳＶ（２株Ⅰ型,３株Ⅱ型）分型结果与病毒分离及血清学方法完全一致。该反应体系与其它来源的ＤＮＡ不产生特异反应,敏感性可达１ｆｇ。应用该法对１５１份临床可疑ＨＳＶ感染的标本进行检测并分型,结果与免疫学方法完全一致。     

样本处理后静置时间对HBV—DNA荧光定量PCR的影响

目的：探讨乙型肝炎病毒核酸（HBV-DNA）荧光定量聚合酶链反应（FQ-PCR）在样本处理后立即加样扩增和4℃冰箱静置24h后再加样扩增对检测结果的影响。方法：68例不同模式乙肝患者样本分别用2种方法处理后进行HBV-DNAFQ-PCR检测。结果：68例样本采用第1种方法共检出HBV-DNA阳性35例,第2种方法共检出39例;第2种方法HBV-DNA阳性定量结果普遍高于第1种方法,指数平均高1个次方左右（u=5.14,P〈0.05）;以第2种方法检测结果为依据,则第1种方法有4例阳性结果漏检,检测结果假阴性率达10.3％（4／39）。结论：样本处理后,应置4℃冰箱静置一定时间,以保证病毒颗粒充分裂解后再进行HBV-DNAFQ-PCR,以提高HBV-DNA的检出率。     

Rapid detection of hepatitis B virus DNA level based on interval-point data fusion of infrared spectra

Hepatitis B is an infectious disease cause by the hepatitis B virus (HBV). In recent years, HBV-DNA level clinically gets more attention for its detailed information than other serological markers. Unfortunately, common clinical method for HBV-DNA level detection is limited for its hours consuming. This study combined infrared spectroscopy with machine learning to investigate the feasibility of near-infrared (NIR) and mid-infrared (MIR) spectra for rapid detection of HBV-DNA level. Based on partial least squares-discriminant analysis (PLS-DA) modeling method, the optimal NIR and MIR models and traditional data fusion models were constructed, respectively. Considering inequal weight between interval and point data in machine learning, interval-point data fusion method was used to compare with other traditional date fusion methods. The results of the study illustrate that interval-point data fusion of NIR and MIR spectra combined with PLS-DA modeling can provide a rapid method for HBV-DNA level detection.     

Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.     

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 μl serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a ³²P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.     

乙肝大蛋白检测在乙肝抗病毒治疗中价值的研究

目的探讨乙型肝炎病毒大蛋白(HBV-LP)在抗病毒治疗过程中的临床诊断价值。方法选取乙型肝炎患者1 000例为研究对象和100例健康体检者为对照组进行HBV-DNA、HBV-LP和ALT检验;然后筛选志愿者进行拉米夫定抗病毒治疗,在治疗前、治疗中和治疗后分别采集血清标本进行HBV-LP、HBV-DNA、ALT检验。结果在HBVM模式中,HBV-LP和HBV-DNA的阳性率分别为42.64%和43.91%(P>0.05)。HBV-LP阳性标本中HBV-DNA、HBeAg和ALT阳性符合率分别为96.27%、65.01%和98.55%,HBV-DNA和ALT优于HBeAg(P<0.05)。志愿者进行拉米夫定抗病毒治疗中HBV-LP与HBV-DNA均下降,HBV-DNA下降更快。结论 HBV-LP可以检测病毒复制、抗病毒疗效观察和反映肝损伤。     

乙肝免疫标志物与乙肝病毒核酸含量与肝功能的关系研究

目的:研究乙肝免疫标记物(HBV-M)与乙肝病毒脱氧核苷酸(HBV-DNA)载量与肝功能的关系。方法:检测104例乙肝患者HBs Ag、HBe Ag、HBc Ab、HBs Ab、HBe Ab5种HBV-M;用PCR法检测HBV-DNA含量;同时检测天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、胆碱酯酶(CHE)等肝功能指标,对三者关系采用Spearman相关性分析。结果:HBs Ag、HBc Ab、HBe Ag阳性组HBV-DNA阳性率及载量均大于其余组(P0.05);HBe Ag含量、ALT浓度与HBV-DNA载量均呈正相关(r=0.48,P0.05)、(r=0.36,P0.05);AST、CHE与HBV-DNA含量无明显相关性,但在无病毒载量与有病毒载量组之间AST比较有统计学意义(P0.05)。结论:HBV-M、HBV-DNA与肝功能之间有一定的相关性,但HBe Ag阴转不代表病毒复制停止,HBV-DNA也不能完全反应肝损害程度,临床应加大重视。     

乙肝患者血清标志物与肝纤维化血清学指标的相关性分析

目的:探讨乙肝患者血清标志物与肝纤维化血清学指标的相关性。方法:收集我院收治的136例乙型病毒肝炎患者,根据轻中重程度分为轻度组、中度组以及重度组,每组42例。观察并比较所有患者乙肝血清标志物、HBV-DNA水平、透明质酸(HA),血清Ⅲ型前胶原肽(PCⅢ),Ⅳ型胶原(CIV)水平以及层粘连蛋白(LN)水平。结果:三组患者的HBV-DNA水平依次升高,与轻度组相比,中度组、重度组患者HBV-DNA水平较高,与中度组患者相比,重度组患者HBV-DNA水平较高,差异具有统计学意义(P0.05)。与轻度组相比,中度组、重度组患者HA、PCⅢ、CIV、LN水平较高,与中度组患者相比,重度组患者HA、PCⅢ水平较高,差异具有统计学意义(P0.05);HBV-DNA水平与HA、PCⅢ、CIV、LN水平均呈显著正相关。结论:乙肝患者的血清学检测指标与血清肝纤维化测定有助于对乙肝的纤维化过程的进展情况进行评估。     

HBV Pre-S1与DNA及其他血清学标志物的相关性研究

目的研究乙型肝炎病毒血清前S1抗原、HBV-DNA与其他乙型肝炎血清标志物HBs-Ag、HBeAg的相关性。方法采用实时聚合酶链反应(Real-Time PCR)技术检测血清中HBV-DNA的含量,用酶联免疫法(ELISA)检测PreS1Ag、HBsAg和HBeAg。采用SPSS 13.0统计软件进行分析,成组设计资料,率比较采用配对的χ2检验,结果的关联性采用独立性的χ2检验。结果以HBV-DNA1.000E+03Copy/ml为阴性组;HBV-DNA≥1.000E+03Copy/ml为阳性组。600例HBsAg阳性的乙型肝炎患者中241例HBV-DNA阳性,283例Pre-S1阳性。其中241例HBV-DNA阳性患者中,Pre-S1阳性222例,阳性率为92.12%。显著高于HBV-DNA阴性组的Pre-S1阳性率(16.99%)。经配对的差异性检验P=0.000(确切概率)。经独立性检验,χ2=326.573,P=0.000。241例HBsAg、HBV-DNA阳性组,HBeAg阳性191例,Pre-S1阳性222例,其中191例HBeAg阳性患者中,Pre-S1Ag阳性185例,阳性率为96.86%。显著高于HBeAg阴性组的Pre-S1Ag阳性率(61.67%)。经配对的差异性检验P=0.000(确切概率)。经独立性检验,χ2=28.511,P=0.000。结论 Pre-S1Ag与HBV-DNA阳性高度相关,有好的一致性和互补性,较HBeAg更敏感。可作为乙肝病毒存在和复制的可靠标志,是反映HBV是否具有传染性的观察指标。     

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.