铕标记基因探针半定量检测丙型肝炎病毒RNA

目的 利用自行设计的引物、探针和新型铕螯合物BHHCT,建立一种半定量丙型肝炎病毒(HCV)RNA的检测方法。方法 收集44份丙型肝炎病人血清,20份正常人血清。利用中山大学达安基因有限公司RNA提取试剂提取HCV RNA,用自行设计的引物进行逆转录聚合酶链反应(RT-PCR)。扩增产物cDNA与微孔板上的捕捉探针杂交后,借助于其上游引物5′端带有的生物素,与标记有铕的链霉亲合素特异性结合,将铕连接到微孔板上,在特定波长激发光激发下,发出荧光,进行检测。结果 铕标记HCV RNA检测法的线性范围为102-106cDNA拷贝,敏感性、特异性均为100％。结论 铕标记RT-PCR检测HCV RNA法的线性范围宽,敏感性、特异性好,检测时间短,无放射性污染,有推广应用的价值。     

混合血浆荧光定量PCR法筛查献血者HCV RNA的初步研究

目的 研究用荧光定量PCR方法检测混合血浆中的HCV RNA。方法 用12人份混合血浆为基本单元提取HCV RNA，逆转录，四份逆转录产物混合上样于HCV PCR扩增反应体系，用PE5700荧光定量PCR仪检测。HCV RNA质控品检测灵敏度。结果 检测4128份标本中1例HCV RNA阳性，HCV RNA冻干质控品检测灵敏度为105IU／ml。结论 48人份混合血浆荧光定量PCR法可用于血液筛查。     

逆转录—半套式—聚合酶链反应单管法检测血清中HCV RNA

目的 探讨建立一种更简便，更特异灵敏的逆转录－聚合酶链反应（RT－PCR）单管法。方法 采用蜡隔将RT和PCR扩增2个独立的反应体系在1个Eppendoff(Ep)管中分隔开来，设计内外引物，优化反应体系及条件，使整个检测反应经高温启动继而连续封闭地完成。结果 在60例血透析患者血清中，33例（55％）抗－HCV－IgG阳性和27例抗－HCV－IgG阴性血清用单管RT－PCR分别检出28例（85％）和3例（9％）HCV RNA阳性，并对3份阳性PCR产物作核酸序列测定。本方法的检测灵敏度为10^2拷贝／ml。结论 RT－PCR单管法在保持高灵敏度的前提下，提高了特异性，具有简单高效，杜绝外源性污染等优点。     

丙型肝炎病毒检测方法的应用评价

目的评价酶联免疫吸附法（ELISA法）检测丙型肝炎病毒核心抗原（HCV—cAg）和抗体（HCV—Ab）以及反转录多聚酶链反应（RT—PCR）检测丙肝RNA,了解三种方法对丙肝病毒检测的优点和缺点。方法采用HCV—cAgELISA试剂盒．HCV—AbELISA试剂盒和丙肝病毒PCR检测试剂盒对来自临床的225例样本进行HCV—cAg、HCV—Ab和HCV—RNA检测。结果225例样本中检出抗HCV阳性28例,其中HCV—cAg阳性14例,RT—PCR阳性16例,剩余的197例抗HCV阴性标本中,检出HCV—cAg阳性3例,其中RT—PCR阳性2例。结论RT—PCR技术检测HCV—RNA是判断丙肝感染的最有效方法。HCV核心抗原检出时间早于抗体,联合运用抗HCV和HCV—cAg或者抗HCV和HCV—RNA检测HCV,能有效降低HCV—Ab检测带来的漏检风险。     

丙型病毒性肝炎的研究现状

丙型病毒性肝炎呈世界分布，而不同人群抗－HCV流行率也不同。经初步调查，北京地区抗－HCV流行率是很高的。血行传播是HCV感染的主要途径。HCV感染50％以慢性过程，约有50％的HCV可演变为慢性肝炎，其中20％转化为肝硬化，57％的胰癌与HCV有关。用EIA、RIA、PCR检测抗－HCV和HCV－RNA，方法为特异性的。丙肝的防治还不理想。     

丙型肝炎病毒抗体联合核酸定量检测对丙型肝炎患者的早期诊断价值研究

目的：探讨在丙型肝炎患者早期诊断中丙型肝炎病毒抗体联合丙型肝炎病毒核酸（HCV RNA）检测的临床价值。方法对116例鼓楼医院住院及门诊丙型肝炎患者采用实时荧光定量聚合酶链反应（FQ-PCR）测定HCV RNA、酶联免疫吸附试验（ELISA）和金标法测 HCV抗体、速率法测丙氨酸氨基转移酶（ALT ）。结果116例丙型肝炎患者中,ELISA HCV抗体阳性97例（占83．6％）,HCV RNA阳性85例（占73．3％）,HCV 金标阳性77例（占66．4％）,ALT阳性69例（占59．5％）,HCV抗体和 HCV RNA联合检测总阳性率（指任-指标阳性即为阳性）为100．0％。结论 HCV抗体和HCV RNA联合检测扩大了丙型肝炎检测范围,降低了丙型肝炎的漏诊率,有利于丙型肝炎的早期诊断。     

应用Tth DNA聚合酶扩增HCV RNA

目的：研究应用Tth DNA聚合酶扩增HCVRNA的意义。方法：35例浙医一院抗HCV抗体阳性病人血清标本中抽提的RNA,在同一管内用Tth DNA聚合酶先后进行RT和第一级PCR扩增HCVC区基因,再按常规进行第二级扩增。结果：35例标本中,用一管法检出29例阳性,用Taq DNA聚合酶的常规RT—PCR法检出27例阳性;两种方法可检出的标本下限浓度分别为10^-4叫和10^-3。结论：用TthDNA聚合酶的一昔法比用Taq DNA聚合酶的常规法敏感性略高．特异性基本相同。     

249例抗—HCV与HCV—RNA检测结果的比较和分析

逆转录-多聚酶链反应（RT－PCR）技术现已较多应用于HCV感染的分子生物学诊断。为了解其与ELISA法检测抗一HCVIN的关系，我们对249例患者同时做此两项检测，作回顾性比较和分析。一、材料和方法HCV－RNA、RT－PCR试剂由本院分子生物实验室制备。检测方法为用30VI血清抽提HCV－R     

树状DNA杂交技术在HCV检测中的应用

目的：建立一种敏感性,特异性,重复性较好的,适同原体群体筛查的检验方法。方法：通过RT－PCR－DDH,RT－nested-PCR和HCV RNA－DDH3种方法检测HCV核酸,推断RT－PCR－DDH技术检测病毒核酸的特异性,敏感性和稳定性。结果：47份ELISA检测HCV抗体阳性血清标本,RT－nested-PCR检出阳性标本33例（70．21％）,RT－PCR－DDH检出阳性标本39例（82．98％）,结论：RT－PCR－DDH技术在逆转录病毒核酸检测的应用中具有较好的特异性,敏感性和稳定性,而且成本较低,操作安全简便,适于中小实验室和基层医院病原微生物的检测。     

建立一种PCR产物直接测序检测HBVYMDD突变的方法

目的 建立检测HBV YMDD变异的PCR产物直接测序法,并与传统实时荧光PCR检测YMDD突变的结果进行比较分析.方法 选取103份慢性乙型肝炎患者血清标本,提取血清HBVDNA.用实时荧光PCR检测YMDD突变;用巢式pcR扩增HBV逆转录酶基因,对PCR产物进行DNA双向测序,NTI软件比对结果.采用Kappa一致性检验对DNA测序法检测的rt204位点突变与实时荧光PCR检测的YMDD突变结果进行比较.结果 PCR产物直接测序法可有效检测低病毒载量标本(500拷贝/ml)和高病毒载量(1010拷贝/ml)标本,同时可以避免高病毒载量标本的抑制效应.与实时荧光PCR相比较,YIDD突变检测符合率为100%,YVDD突变检测符合率97.1%,YIDD与YVDD共生突变符合率76.2%(Kappa=0.853,P<0.01).结论 本研究建立的PCR产物直接测序法检测HBV耐药相关基因突变,灵敏度高,检测范围宽,与实时荧光PCR检测结果有较高的符合率,并可同时检出YMDD、YIDD和YVDD.     

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.     

Cell assay using a two-photon-excited europium chelate

We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu(3+) emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles.     

逆转录-环介导等温扩增快速检测EV71病毒

目的建立一种检测肠道病毒71型（EV71）快速、敏感的一步逆转录-环介导等温扩增（RT-LAMP）方法。方法针对EV71病毒VP2基因特异性序列的6个区域设计4条LAMP引物,建立RT-LAMP检测方法,并评价其特异性和灵敏度。结果通过GoldView染色和凝胶电泳均能观察到LAMP扩增产物的存在,且与柯萨奇病毒A16型（CA16）无交叉反应发生。所建立的RT-LAMP检测方法灵敏,最低检测限为1.0×102copies/mL。RT-LAMP检测的41份咽拭子标本中有27份出现EV71阳性反应,与荧光定量PCR结果一致。结论 RT-LAMP是一种快速、敏感、特异、准确的方法,适合用于基层医疗机构临床检测。     

Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology.

BACKGROUND: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. METHODS: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 x 10(10) L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. RESULTS: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2-10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). CONCLUSIONS: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.     

Digoxygenin-labelled DNA-probe: a rapid non-radioactive method for hepatitis B virus DNA detection in serum.

The sensitivity and specificity of two non-radioactive spot hybridization assays for hepatitis B virus DNA (HBV-DNA) using biotin and digoxygenin-labelled DNA probes were investigated in parallel in 122 serum samples from patients with chronic hepatitis B and 50 controls. The results were compared with an isotopic technique using a 32P-labelled probe. HBV-DNA was detected in 56 (80%) out of 70 hepatitis B "e" antigen (HBeAg)-positive cases and in 4 (8%) out of 52 antibody to hepatitis B "e" antigen (anti-HBe)-positive cases using the digoxygenin or 32P-labelled probes. No false positives were found with either method. Using the biotin-labelled probe, 16% of sera gave discordant results, which were considered to be false positive. The time required for detection of serum HBV-DNA was 2 hours for the non-radioactive probes and 16 hours for the isotopic probes. This study suggests that the digoxygenin-labelled probe for detection of HBV-DNA is the most rapid and sensitive method for routine diagnosis of viral replication in clinical laboratories.     

连接酶链反应-酶联免疫吸附试验诊断沙眼衣原体感染的初步应用

目的 建立连接酶链反应-酶联免疫吸附试验(LCR-ELISA)检测沙眼衣原体DNA的方法,并初步应用于检测新生儿沙眼衣原体感染。方法 针对沙眼衣原体外膜蛋白基因设计4条引物,并分别在其两端标记地高辛和生物素。用此4条引物进行LCR扩增沙眼衣原体DNA。带有生物素和地高辛双标记的扩增产物以ELISA方法显色判断结果。采集328份肺炎新生儿鼻咽标本,分别对其进行培养和LCR扩增,应用ELISA检测有无沙眼衣原体感染。结果 LCR-ELISA方法可以检测出6种沙眼衣原体标准株,而对2种肺炎衣原体和其他细菌DNA无反应,显示出较强的特异性;可检测出10fg的DNA,较传统电泳法敏感10倍,且避免了溴化乙啶的污染。328份标本中,LCR-ELISA检测出68份阳性,培养仅检测出60份,两者相比差异有显著性。以扩大的金标准判断,LCR-ELISA的特异性、敏感性分别为100％和98.6％;而培养的特异性、敏感性分别为100％和、86.9％。结论LCR-ELISA是一种特异而敏感的基因扩增方法,避免了溴化乙啶污染,判断结果客观,检测方便。经临床初步应用,取得良好结果,值得进一步研究和完善。