57例抗HIV初筛阳性确认实验带型分析

目的 调查HIV感染初筛实验的准确率及确认抗HIV初筛试验阳性血清的感染和感染型别。方法 调查我国艾滋病高发农村，对部分初筛阳性和可疑的血清进行确认实验。结果 57例初筛为抗HIV阳性和可疑血清进行确认实验，56份为HIV—l阳性，其中1份样本出现HIV—2型反应条带；1份阴性。确认阳性标本的反应条带中，抗外膜蛋白(env)抗体阳性率最高，gpl60为100％，gpl20为94．6％，gp41为91．1％；多聚酶抗原(pol)p66为82．1％；核心抗原(gag)P24为53．6％。提示无症状携带组确认反应条带的pol和gag的阳性率显著高于艾滋病组(P＜0．01)。儿童组gag阳性率显著低于成人组(P＜0．05)。结论 HIV感染初筛实验的准确率较高，但确定感染需靠确认实验。外膜蛋白、p66和p24抗原是HIV感染的重要抗原，对确认HIV的感染具有指导意义。     

人免疫缺陷病毒Ⅰ型gag、env、rev mRNA检测

目的动态检测人免疫缺陷病毒Ⅰ型(HIV-1)基因gag、env、rev mRNA水平.方法将插入有HIV-1竞争模板的pSPI质粒DNA转化入感受态菌STBL-2,进行扩增.以T7 RNA聚合酶体外转录出HIV-1 mRNA竞争模板.用3对引物对HIV-1 RNA标本进行定量竞争聚合酶链反应(QC-RT-PCR), 检测HIV-1 gag、env、rev mRNA 水平.结果 HIV-1 ⅢB感染的标本中,HIV-1 gag、env和rev mRNA的水平分别为36 000拷贝/μl,9 800拷贝/μl和9 100拷贝/μl.此方法可动态定量检测HIV-1 gag、env、rev mRNA水平.结论该方法简便易行,费用低廉,适用于实验室HIV致病机理、药物筛选和病毒复制状况的研究.     

液相芯片法验证蛋白质免疫印迹试验检测HIV-1结果的敏感性和特异性

目的初步分析采用液相芯片法研制人类免疫缺陷病毒(HIV)-1检测试剂的可行性。方法采用液相芯片技术用蛋白质免疫印迹试验(Western Blot)检测HIV-1阳性和阴性的标本;用HIV-1的gp120、gp41、p24抗原分别包被不同编号的免疫磁珠,与生物素化二抗、亲和素化荧光染料PE组成液相芯片检测系统,对标本进行检测分析。结果 55份标本经Western Blot法确认阳性样本检测符合率为100%;76份经酶联免疫吸附试验(ELISA)初检和复检阴性的标本检测HIV-1gp120、HIV-1gp41、HIV-1p24抗原均为阴性;86份经ELISA法检测初检和复检为阳性而Western Blot法确认为阴性的样本,其HIV-1gp120、HIV-1gp41、HIV-1p24单片段检测均为阴性。液相芯片法检测HIV-1抗体的敏感度和特异度均为100%。结论采用液相芯片技术组成的HIV-1抗体检测系统,其敏感度和特异度均优于ELISA法检测。     

用邻位相连法方法确定HIV-1M组基因分型的最佳区域

目的为进一步确定HIV-1M组基因分型的最佳区域。方法从Genbank中筛选出对各成熟肽区域有注释来源,来自于不同国家地区的104条HIV-1M组全基因组序列。对序列的结构区域gag区、pol区、env区及env的亚区域gp41和gpl20分别建邻位相连（NJ）系统进化树。结果pol区未能把D亚型和B亚型分开;gag区未把K亚型和F亚型分开;env区能对HIV-1M组正确分型;env的亚区域gp41未能把K亚型与H和F亚型分开,J亚型未与A亚型分开。env的亚区域gpl20未能把J亚型与A1亚型分开。结论用NJ系统进化树方法确定enV区最能代表HIV-1M全基因组序列进行分型。     

干血斑样本用于人类免疫缺陷病毒1型前病毒基因诊断的研究

目的研究干血斑（DBS）样本用于人类免疫缺陷病毒1型（HIV-1）感染前病毒DNA基因诊断的可行性。方法采集59例静脉吸毒HIV-1感染者和22名HIV-1阴性健康人EDTA抗凝静脉全血5ml，保存全血1ml，另用50μl制备干血斑样本后，分离血浆。QIAamp Blood Mini试剂盒和10％Chelex 100树脂分别提取全血和干血斑中细胞DNA。Roche Cobas Amplicor逆转录聚合酶链反应检测HIV-1感染者血浆病毒载量。HIV-1Env基因Gp41区，gag基因，pol基因分别用2对外侧和内侧引物，按照套式PCR方法，进行扩增。以HIV-1任何两个基因区扩增结果组合判断HIV-1感染。结果在重复2～3次的前提下，59份DBS样本HIV-1感染基因诊断敏感度93％（95％可信区间89％～97％），34份全血样本94％（95％可信区间89％～98％）。疋。检验显示，两种样本用于HIV-1基因诊断时差异无统计学意义。22份两种阴性样本特异性均为100％（95％可信区间95．93％～100％）。8份血浆病毒载量（VL）〉4．0 Log的DBS样本用于基因诊断的可重复性和HIV-1感染样本检出率优于5份VL〈4．0 Log样本。18对全血和DBS样本HIV-1基因诊断一致性分析，符合率94％，一份DBS样本HIV-1特异三个基因片段均未扩增。结论干血斑样本易采集，便于运输，保存条件低，费用低廉，用于HIV-1基因诊断时与全血样本检测结果无显著性差异，病毒载量〈4．0 Log的样本，为增加检出概率需要对样本重复检测。DBS样本HIV-1基因诊断方法适合于无条件立即处理血样本的偏远地区或采样困难的感染者的诊断，及HIV-1母婴垂直传播研究中婴幼儿感染的早期诊断。     

人免疫缺陷病毒I型gag、env、rev mRNA检测

目的：动态检测人免疫缺陷病毒I型（HIV－1）基因gag,env,rev mRNA 水平。方法：将插入有HIV－1竞争模板的pSPI质粒DNA转化入感受态菌STBL－2，进行扩增，以T7RNA聚合酶体外转录出HIV－1 mRNA竞争模板，用3对引物对HIV－1 RNA标本进行定量竞争聚合酶链反应（QC－RT－PCR），检测HIV－1 gag,env,rev mRNA水平。结果：HIV－1 Ⅲ B感染的标本中，HIV－1,gag,env和rev mRNA的水平分别为36000拷贝／μl，9800拷贝／μl和9100拷贝／μl，此方法可动态定量检测HIV－1 gag,env,rev mRNA水平。结论：该方法简便易行，费用低廉，适用于实验室HIV致病机理，药物筛选和病毒复制状况的研究。     

HIV1＋2型抗体化学发光免疫分析试剂盒的研制

[目的]建立：险测HIV1＋2型抗体化学发光免疫分析方法并研制其试剂盒。[方法]应用HIV-1的gp41和gp120．HIV-2的gp36蛋白混合包被化学发光微孔板作为固相抗原；用辣根过氧化物酶（HRP）标记以上抗原，应用含Luminol及其增强剂的化学发光底物作示踪剂，通过条件优化建立检测血清中HIV-1＋2型抗体的双抗原夹心化学发光免疫分析法。应用该方法制备HIV1＋2抗体化学发光免疫分析试剂盒三批，分别检测中国药品生物制品检定所（中检所）HIV抗体国家参考品，并与国内市场主要产品进行对比。[结果]建立了HIV1＋2型抗体化学发光免疫分析试剂盒（双抗原夹心法）。用中检所HIV抗体国家参考品检测，该方法研制的三批试剂盒均符合质量标准。与国内知名厂家的HIV-ELISA试剂盒比较，该方法所研制的试剂盒在灵敏度和精密性上较好。检测196份HIV1／2感染血清，226份其他疾病患者血清和正常人血清，阳性和阴性符合率均100％，与HIV-ELISA试剂相关性达0．994。三批变异系数均小于10％。试剂盒于37℃放置6d后，检测结果的阴阳性判断不受影响。[结论]该法特异性强，灵敏度高，精密性及稳定性好，适用于献血员的筛查和临床HIV感染的检测。     

HIV-1跨膜蛋白gp41重组抗原的表达及其免疫反应性

目的 为开发和建立敏感、特异的抗HIV-1抗体的检测方法研制重组gp41抗原.方法 根据HIV-1的基因序列,设计并合成了1对PCR扩增引物,应用PCR技术从HIV-1 外膜基因组中扩增出HIV-1的gp41截短体,将扩增的基因片段插入质粒pET-28a中构建成重组表达质粒pET-gp41.诱导表达并纯化gp41重组抗原,对gp41进行了初步应用分析.结果 gp41在大肠埃希菌BL21 (DE3)中获得高表达,表达量占菌体总蛋白量的26.08%.纯化后截短体gp41的纯度为97.94%.经间接ELISA和免疫印迹检测,纯化后的表达产物gp41具有很高的抗原特异性和免疫反应性.结论 研制的重组抗原gp41有较强的抗原性和潜在的应用价值.     

HIV-1/2/O 抗体联合诊断试剂盒的制备及其检测效果的研究

目的 制备HIV-1/2/O抗体检测试剂盒并评估其检测效果.方法 采用RT-PCR分别获取HIV-1型M、O组的gp41以及HIV-2 gp36的优势表位基因,并通过柔性链将各表位基因嵌合,重组表达,亲和纯化、辣根过氧化酶标记,与包被抗原配对筛选,获取检测HIV 抗体的双抗原试剂.结果 成功嵌合各表位基因并获得表达,纯...     

北京市一例HIV-1 CRFl5_01B亚型类似毒株的鉴定

目的 通过对一例HIV-1毒株env、gag和pol基因的序列分析,表明HIV-1 CRF15_01B亚型类似毒株已在北京市出现.方法 从一例在北京居住的四川籍女性HIV-l感染者(BJ06108,通过性途径感染)血浆中提取病毒RNA,使用套式PCR方法扩增env、gag和pol基因,并进行序列测定和亚型分析.结果 病例BJ06108在env、gag和pol基因区与CRF15_01B亚型参考株1501B.TH.99.99TH_MU2079基因离散率较近,基因离散率分别为8.8%、6.1%和7.2%.系统进化树分析表明BJ06108与CRF15_01B亚型参考株1501B.TH.99.99TH_MU2079聚在一起.结论 CRF15_01B亚型类似毒株已在北京市出现,应该加强HIV-1毒株亚型变异的监测.     

浙江省绍兴市46例男男性行为者HIV感染基因特征分析及流行病相关因素分析

目的 了解浙江省绍兴市男男性行为者(MSM)人群中HIV-1感染者的亚型类型和特征 。方法 分析2013年1月至2014年7月本地区新确证未经抗病毒治疗的50例MSM感染者样本,采用RT-PCR和nest-PCR方法扩增HIV-1的gag和pol全长基因区,成功扩增46份样品,构建系统进化树分析基因亚型。结果 46份样品中发现CRF01_AE、CRF07_BC和B三种亚型和1株AE、BC重组亚型,各占52.17%、39.13%、6.52%和2.17%。结论 绍兴市 HIV-1感染的MSM人群中主要流行的亚型为CRF01_AE和CRF07_BC,该人群感染率高,应加强监测。     

PCR和巢式PCR直接检测血清中HIV核酸序列

采用ＰＣＲ和巢式ＰＣＲ方法对５３例ＨＩＶ抗体阳性和阴性标本进行了检测，以扩增ＨＩＶ特异性ＤＮＡ序列，结果美国ＡｂｂｏｔｔＨＩＶ阳性血清２份，新加坡阳性血清２份，国内阳性血清２份及ＨＩＶ－１病毒培养物１份，两套ＰＣＲ扩增反应物均为阳性，对其中一套ＰＣＲ扩增的ｇａｇ基因序列进行巢式ＰＣＲ也为阳性。采自西南边境地区的２３份ＥＬＩＳＡ和ＷＢ阳性血清，各种ＰＣＲ反应均为阳性；１１份ＥＬＩＳＡ可疑阳性、ＷＢ阴性标本中，有两例血清呈ＰＣＲ阳性，探针杂交亦为阳性。而１２例ＳＩＶ、ＳＲＶ及猴血清标本ＰＣＲ反应均为阴性。上述结果表明，采用ＰＣＲ方法检测血清中ＨＩＶ核酸序列是一种很有前途的ＨＩＶ感染诊断方法。     

Human immunodeficiency virus (HIV) nef-specific cytotoxic T lymphocytes in noninfected heterosexual contact of HIV-infected patients.

We report on the detection of HIV-specific cytotoxic T lymphocytes (CTL) among 23 regular partners of HIV-infected individuals. 15 of the 46 individuals enrolled in the study were positive for HLA-A2.1 typing. Among the 23 contacts studied, 7 were seropositive and 16 were seronegative on repeated tests. None of the 16 seronegative contacts were positive for p24 antigenemia nor were they positive by the lymphocytes coculture assay, although, in two instances HIV-1 DNA could be detected by PCR (in one case using a gag SK 38/39 primer, and in the other using a primer for the pol P3/P4 primer). These two individuals remained seronegative for 18 and 36 mo, respectively. HIV-specific cytotoxicity was performed in the 15 HLA-A2.1 subjects (7 indexes, 2 seropositive contacts, and 6 seronegative contacts) and in 4 HLA-matched HIV negative donors. CTL specific for env, gag, or nef proteins could not be detected in unstimulated bulk cultures of peripheral blood lymphocytes in any of the six seronegative contacts. However, using a limiting dilution assay we found an usually high frequency of HIV nef-specific CTL precursors (CTLp) for HIV env and gag was very similar to that observed in seronegative HLA-matched healthy donors. Because no presence of HIV could be demonstrated in these individuals, these findings argue against the possibility of a silent HIV infection and suggest that a CTL response against nef may be involved in a rapid and effective clearance of the virus after sexual exposure.     

An optimized nested polymerase chain reaction (PCR) approach allows detection and characterization of human immunodeficiency virus type 1 (HIV-1) env and gag genes from clinical samples

The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1 fg and 100 fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 10(8) and 10(4) of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples.     

Detection of HIV-1 infections by PCR: evaluation in a seropositive subject population

To study the polymerase chain reaction (PCR) performance in detecting human immunodeficiency virus (HIV) infections, we tested 53 HIV-1 seropositive patients and 29 HIV-1 seronegative subjects for four different HIV-1 DNA regions. Fifty-one seropositive patients were found positive by PCR with at least one primer pair, but two were repeatedly negative for all primers. Weekly blood samples from 12 seropositive subjects all detected positive for at least one primer pair, but for three patients an irregular primer detection pattern was found. One additional HIV-1 seropositive sample, found negative for HIV DNA, was also negative for the beta-globin PCR control. The 29 seronegative specimens were HIV-1 DNA negative, as was a HIV-2 seropositive patient. This study demonstrates that PCR is almost as good as serological tests for detecting HIV infections, with a specificity of 100% and a sensitivity of 96% and that resampling the patients may improve detection performance.     

Sensitive non-radioactive detection of HIV-1: use of nested primers for the amplification of HIV DNA.

This report describes the use of the polymerase chain reaction (PCR) for the non-radioactive detection of HIV-1 proviral genomic sequences in HIV-1 infected cells. We have developed a sensitive assay, using three different sets of nested primers and our results show that this method is superior to standard PCR for the detection of HIV-1 DNA. The assay described features the use of a simple and inexpensive sample preparation technique and a non-radioactive hybridization procedure for confirmation of results. To test the suitability of the assay for clinical purposes, we tested cell samples from 76 anti-HIV-1 positive patients. All were positive for at least one primer set: 88% were positive for all three sets of primers; 9% were positive for two sets of primers and 3% were positive for only one set of primers. It provides a useful approach to the study of HIV-1 infection in patient samples where genomic copies often are present at such low numbers that they are otherwise undetectable.