一种初步快速甄别总肠道病毒的基因分型方法

目的 建立并验证总肠道病毒5’-非编码区(5＇-non coding region,5＇-NCR)PCR扩增测序并在NCBI数据库比对进行生物信息分析的方法,为手足口病的早期快速诊断提供敏感、高效的总肠道病毒分子分型检测方法. 方法 收集165例临床检测阳性的标本,提取总RNA扩增人肠道病毒A、B、C和D型5＇端非编码区用于总肠道病毒初步基因分型,PCR产物测序并在Genbank中进行BLAST分析.结果 成功从临床咽拭子标本中扩增了5＇-NCR用于基因分型.165例总肠道病毒阳性标本中鉴定出12种肠道病毒类型,有77例人肠道病毒(enterovirus type 71,EV-71),37例人柯萨奇病毒(coxsackievirus,CV)CV-A16,31例CV-A4,20例其它型别. 结论 5＇-NCR PCR测序是总肠道病毒基因分型和监测的敏感、简单、早期、快速的检测方法.     

人类免疫缺陷病毒1型感染基因诊断方法的建立

目的 建立人类免疫缺陷病毒1型(HIV-1)基因诊断方法。方法 随机选取80份全国送检的已确认的HIV阳性样品，从健康献血员中选取40份HIV阴性样品。分别提取人DNA和病毒RNA，再分别扩增HIV-1的gp41、pol和gag各基因区的3套反应系统，进行巢氏聚合酶链反应(nPCR)和逆转录聚合酶链反应(RT-PCR)。将结果与血清学方法比较，观察结果是否一致。同时检测3套反应系统的敏感性、重复性和对HIV-1各亚型参考病毒株的扩增情况。结果 用3套反应系统对80份阳性样本进行nPCR检测的阳性率为：gp41反应系统88．8％；pol反应系统83．8%；gag反应系统81．3％；3套系统联合应用检出率90.0％。用RT-PCR检测阳性率为：gp41反应系统96．3％；pol反应系统91．3％；gag反应系统95．0％；3套反应系统联合应用检出率可达98．8％。阴性样品扩增均阴性，特异性为100％。3套反应系统不但可扩增HIV-1型M组的A、B、C、D、CRF01-AE、F、G、H亚型，还可扩增其N和O组毒株，且与HIV-2无交叉反应。nPCR最低检测限为1拷贝／PCR。gag和pol反应系统RT-PCR最低检测限为750拷贝／ml；gp41反应系统最低检测限为150拷贝／ml。nPCR和RT一：PCR扩增gp41、pol和gag基因区的重复性分别为：93．3％、90．0％、86．70h，和100%、96．7％、100％。结论 建立的HIV-1基因诊断方法敏感性、特异性和重复性较好，且成本低廉，适合我国HIV检测实验室应用。     

人类免疫缺陷病毒检验技术研究进展

人类免疫缺陷病毒(HIV)是引起获得性免疫缺陷综合征和相关疾病的RNA病毒,主要侵犯CD4T细胞、CD4单核细胞和B淋巴细胞。自从人类发现第一例艾滋病患者后,HIV感染者越来越多,据统计,到2004年底,全球有四千万人感染HIV,其中有280～350万人死于艾滋病。最严重的是南     

鲍曼不动杆菌分子流行病学的基因分型技术及应用

鲍曼不动杆菌(Acinetobacter baumannii,AB)已成为医院感染最重要的病原菌之一,对AB菌所致感染的流行病学研究具有重要意义。传统的细菌流行病学分型方法由于分辨率低、影响因素多、重复性较差,在医院感染监测与预防控制中的应用日益减少,而基因分型技术则逐渐成为临床上监测和鉴定细菌的主要手段。本文对国内AB菌分子流行病学研究中常用的基因分型技术及应用现状作一综述。     

乙型肝炎病毒的基因分型

乙型肝炎病毒(HBV)基因型是HBV基因异质性的表现之一,最早于1988年提出,至今已发现的基因型有A~H共8种。研究发现HBV基因型在不同地域和人群中的流行谱有差异,这种差异将影响HBV各种检测方法在不同地区和人群中的检测准确性。同时,HBV基因型还与乙型肝炎病情的进展、预后以及对治疗的应答有一定相关关系。因此,HBV的基因分型研究对进一步明确乙型肝炎发病机制、提高检测效率、寻找治疗对策和实现流行病学控制具有重要意义。一、HBV基因分型的历史回顾1988年,Okamoto等[1]对采自日本和印度尼西亚的3名无症状HBV携带者血清型为adw…     

乙型肝炎病毒的基因分型

乙型肝炎病毒（HBV）基因型是HBV基因异质性的表现之一，最早于1988年提出，至今已发现的基因型有A～H共8种。研究发现HBV基因型在不同地域和人群中的流行谱有差异，这种差异将影响HBV各种检测方法在不同地区和人群中的检测准确性。同时，HBV基因型还与乙型肝炎病情的进展、预后以及对治疗的应答有一定相关关系。     

霍乱弧菌的分子分型方法

在霍乱流行和暴发调查中,追溯传染源和调查传播途径往往需要对霍乱弧菌进行分型分析.对霍乱弧菌进行血清分型和噬菌体一生物分型,是得到菌株基本信息不可缺少的手段.如果想要揭示菌株在分子水平上的变异和进化规律,则需要进行分子分型分析.本研究对霍乱弧菌的各种分子分型方法进行逐一介绍并加以综合比较.     

反向斑点杂交-基因芯片检测人类乳头瘤病毒及分型

目的 检测人类乳头瘤病毒(human papilloma virus,HPV)及其分型,了解其在女性宫颈中的感染情况与宫颈癌防治方面的意义.方法 采用反向斑点杂交-基因芯片检测220例不同年龄患者宫颈中HPV感染情况与分型情况,对比分析低危型与高危型HPV亚型感染率以及其与年龄的关系.结果 220例标本中HPV感染阳性...     

艾滋病合并淋巴瘤患者抗人类免疫缺陷病毒治疗后短期内出现病毒耐药1例

<正>艾滋病(AIDS)是由于感染人类免疫缺陷病毒(HIV)导致的严重威胁人类生命健康的疾病。AIDS相关淋巴瘤(ARL)是AIDS患者最常见的机会性肿瘤，也是导致死亡的主要原因之一^[1]。ARL分很多种类，95%为B细胞来源，且超半数为中高度恶性淋巴瘤，主要以弥漫性大B细胞淋巴瘤为主^[2-3]。化疗期间联合抗反转录病毒治疗(ART)可提高完全应答率。     

汉赛巴尔通体的分子分型研究进展

汉赛巴尔通体是一种新发的人兽共患病病原,呈全球性分布。随着分子生物学技术的发展,多种分子分型方法应用于其流行病学调查,加深了研究者们对汉赛巴尔通体种内变异和系统发育关系的了解。本文就几种常见的分子分型方法在汉赛巴尔通体分子流行病学研究中的应用做一综述。     

生殖支原体环介导等温扩增检测方法的建立

目的建立检测生殖支原体(M.genitalium,Mg)的环介导等温扩增方法。方法根据Gen Bank公布的Mg核苷酸序列,设计4条特异性引物,进行PCR反应,对内、外引物浓度,d NTPs和Mg SO4浓度及Bst DNA聚合酶用量等进行优化,建立Mg LAMP的检测方法并进行特异性和敏感性评价;对20份HIV感染者的DNA样本分别进行LAMP和常规PCR检测。结果内、外引物浓度分别为1.6和0.2μmol/L,d NTPs浓度为1.6 mmol/L,Mg SO4浓度为12 mmol/L,Bst DNA聚合酶用量为8 U时,LAMP反应效果较好;用该体系检测同源关系较近的穿通支原体(M.penetrans,Mpe)、梨支原体(M.pirum,Mpi)均呈阴性;对Mg的检测灵敏度可达10-7;20份HIV感染者用LAMP法及常规PCR法各检出Mg阳性标本5份,两法的检测结果一致。结论建立的Mg LAMP检测方法的特异性、灵敏度较好,成本低廉,适合临床应用。     

Xpert HIV-1病毒载量检测系统用于HIV-1病毒载量检测的临床价值

目的探讨Xpert HIV-1病毒载量(Xpert HIV-1 VL)检测用于HIV-1病毒载量检测的临床价值。方法采用Xpert HIV-1 VL和Abbott m2000 Real Time检测系统(Abbott m2000)同时检测191份血浆样本的HIV-1病毒载量。采用符合率、卡方检验和Kappa值等统计分析两种检测方法定性检测结果的一致性,采用相关性、回归分析和Bland-Altman模型等统计分析两种检测方法定量检测结果的一致性。结果两种检测方法的阳性符合率、阴性符合率和总符合率均为100%,两种方法检测结果一致性分析的Kappa值为1.00(P<0.001);两种方法检测结果的相关系数为0.9576,回归分析表现出较高的相关性(R2=0.9170),Bland-Altman模型分析显示两种方法检测有95.3%(164/172)的样本在95%一致性界限内。结论Xpert HIV-1 VL用于检测HIV-1 RNA具有很高的灵敏度、准确性和稳定性,且操作简单、方便、安全,适合临床用于对HIV-1患者血浆样本中HIV-1 RNA的检测。     

A solid phase plate assay for HIV-1 genotyping

A prototype solid phase plate assay (SPPA) for the detection and genotyping of HIV-1 subtypes was developed using a PCR-based capture hybridization format. Well characterized HIV-1 reference controls of known subtypes and subtype specific capture oligonucleotide probes targeting several regions of the envelope (env) gene of HIV-1 were selected to develop the assay. The subtype specific oligonucleotide probes were covalently bound to microtubes in an ordered pattern and biotin labelled primers were used to amplify the target sequences. The PCR products were hybridized to the corresponding oligonucleotide probes, and colorimetrically detected by a chromogenic reaction using a standard microplate reader. All the HIV-1 subtype reference controls specifically hybridized to the corresponding capture probes and negligible cross-hybridization between subtypes was observed. To demonstrate the performance and reproducibility of the SPPA system and its validation with clinical samples, several human plasma samples of unknown and known HIV-1 subtypes were tested. The SPPA is highly specific and unambiguously identify the major subtypes of the HIV-1 M and O groups. This assay could be a useful method for subtyping samples of HIV-1 infected individuals and for disease management.     

1型HIV感染多重PCR检测方法的建立

重复性,能覆盖我国最主要的流行病毒株.     

Urinary HIV-1 antibody patterns by western blot assay.

Diagnosis of human immunodeficiency virus infection (HIV-1) is normally carried out by serum testing for HIV-1 antibody. Recently, antibody testing in other body fluids such as saliva and urine have been attempted. In this study, we examined HIV-1 antibody patterns in urine by Western blot assay as compared to that found in serum. Out of 44 sero-positive samples by Western blot assay we found 43 to be HIV-1 antibody positive in the urine, whereas all 40 sero-negative samples were negative in urine. Thus the sensitivity of urine testing was 97.7% with 100% specificity when compared to serum testing by the Western blot assay. In the analysis of the antibody pattern in urine, we found 6.8% of p17, 68% of p24, and 47.7% of p39 in the core proteins; 72.7% of p31, 61.4% of p51, and 68.2% of p66 in the polymerase; and 63.6% of gp41, 75% of gp120, and 97.7% of gp160 in the envelope proteins. The data obtained supports the selection of the HIV-1 antigen subtype-E to develop a home test kit using urine. Urine testing for HIV-1 antibody is convenient, non-invasive, safe, and easily performed at home. However, if the urine is positive, the confirmation test on serum is needed.     

Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.     

High-throughput human immunodeficiency virus type 1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target

Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable Z' value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >10(6) compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.