检测HIV—1载量的荧光实时定量PCR技术的建立其应用

准确测定HIV－1的前病毒载量和病毒载量的技术,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面,都具有十分重要的应用价值。以定量的HIV－1 DNA和RNA为标准外参照,利用SYBR Green荧光染料和GeneAmp5700Sequence Detection System（5700系统）,建立了测定HIV－1的前病毒载量和病毒载量的荧光实时定量PCR技术,以病毒感染细胞和培养上清上材料,测定了三种化合物（AZT,GL和WT）对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较,根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小,提出以荧光实时定量PCR技术测定前病毒载量,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV－1前病毒方面有特别的价值。     

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

准确测定HIV-1的前病毒载量和病毒载量的技术,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面,都具有十分重要的应用价值。以定量的HIV-1DNA和RNA为标准外参照,利用SYBRGreen荧光染料和GeneAmp5700 Sequence Detection System(5700系统),建立了测定HIV-1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料,测定了三种化合物(AZT,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小,提出以荧光实时定量PCR技术测定前病毒载量,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV-1前病毒方面有特别的价值。     

SYBR GreenⅠ实时荧光定量RT-PCR测定肠道病毒71型（EV71）RNA拷贝数方法的建立

目的建立SYBR GreenⅠ荧光染料实时定量RT-PCR方法,测定实验动物等来源的EV71病毒RNA。方法运用EV71VP1保守区引物,优化real time RT-PCR条件,运用NASBA方法扩增EV71病毒RNA,计算拷贝数,经10倍系列稀释做出标准曲线,作为EV71病毒RNA定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR Green RT-PCR Kit,该标准品可精确定量到100copies/μL,PCR扩增效率达到99.5%。结论 SYBRGreenⅠ荧光染料实时定量PCR法测定EV71病毒RNA拷贝数的方法敏感性高、稳定性好,可用于EV71病毒RNA载量的定量测定。     

猴免疫缺陷病毒（SIV）在艾滋病模型恒河猴组织中的分布和载量测定

目的研究当艾滋病恒河猴模型的血浆病毒载量处于低水平或阴性时,猴免疫缺陷病毒（simian immunodeficiency viruses,SIV）在宿主组织中的分布情况。方法SIVmac251感染恒河猴10只,定期检测其血浆载量,感染病毒平均高峰时间第14天时,活检取淋巴结。选取感染18个月后病毒载量最低水平和阴性的2只艾滋病猴（SAIDS）,经安死术后取淋巴结、脾、肝、肺、肾、脑等组织,用原位杂交和实时荧光定量PCR的方法检测病毒在组织中的分布和组织中的病毒载量。结果感染后14d,10只猴血浆病毒载量达到10^7copies/mL,淋巴结组织病毒载量为10^5-10^8copies/g,原位杂交方法在腹股沟淋巴结中检测到强阳性斑点。感染后第18个月的2只猴,血浆病毒载量下降并维持不高于10^2copies/mL水平或阴性,但组织分布不尽相同,在肠系膜淋巴结、肾上腺、海马回、空肠、脾脏等组织中检测到10^5-10^6copies/g的病毒载量,于一只猴的脑积液中检测到10^3copies/mL的病毒载量。用原位杂交的方法在肠系膜淋巴结和空肠中检测到强阳性斑点,其它组织中未检测到阳性斑点。结论实验证实SAIDS猴在血浆病毒载量低甚至阴性时,病毒在不同组织中仍有分布,有些组织中甚至出现高病毒载量,提示在制备SIV/SAIDS模型中,尤其在药物筛选和疫苗评价时,应考虑组织病毒载量指标的测定和药物、疫苗对组织病毒的治疗清除作用的评价。     

大连市HIV感染者/艾滋病患者在抗病毒治疗过程中病毒载量及细胞免疫指标变化的相关性分析

目的研究大连市HIV感染者/艾滋病患者抗病毒治疗过程中,HIV病毒载量和细胞免疫指标的变化情况。方法选取2013年开始抗病毒治疗,治疗时间满一年的HIV/AIDS 60例,分别在进行抗病毒治疗过程中的0、6、12月用全自动病毒载量分析仪(COBAS AmpliPrep/COBAS TaqMan)检测HIV病毒载量,同时用流式细胞计数仪(FACSCalibur)检测CD4~+和CD8+T淋巴细胞数。结果60例HIV/AIDS中治疗前仅有9例患者有检测数据,都是高病毒载量(VL1 000),在治疗6个月后,病毒载量抑制比例为73.33%(44/60)、治疗12个月后,载量抑制比为81.67%(49/60)。两组治疗后比治疗前病毒载量抑制比显著升高(Z=12.85,P0.001;Z=16.35,P0.001)。随着治疗时间的推移,CD4~+T淋巴细胞数和CD4~+/CD8~+比值都有所上升,差异有显著性(F=72.73,P0.001;F=53.83,P0.001)。病毒载量越低,T淋巴细胞数及CD4~+/CD8~+比值越高,呈负相关,有显著性差异(F=21.66,P0.001;F=8.53,P0.001)。结论抗病毒治疗有助于提高CD4~+T淋巴细胞数,改善HIV/AIDS免疫状态。细胞免疫指标与病毒载量的相关性呈负相关。     

应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率

慢病毒载体已经广泛应用于动物模型中基因治疗的研究和转基因动物的制备．而准确地测定重组慢病毒的滴度和感染效率是其关键步骤．通过荧光实时定量PCR的方法定量分析重组慢病毒的颗粒数以及病毒的活性滴度,并以GFP报告基因的方法作为对照来验证定量PCR方法的准确性．研究结果显示,应用荧光实时定量PCR法与GFP报告基因法测定得到的病毒活性滴度成正相关,而且前者可以更加准确地测定病毒滴度和病毒感染效率．     

季节性流感病毒H1N1实时荧光定量PCR检测方法的建立

目的建立一种快速定量检测季节性流感病毒H1N1核酸的实时荧光定量PCR检测方法及试剂盒。方法选择季节性流感病毒H1N1的保守基因NP基因作为检测靶目标,应用Clustal W软件进行序列同源性比对分析,筛选出季节性流感病毒H1N1特异性的保守序列作为引物候选区域,然后应用Primer Express及PrimerPremier 5.0软件包对候选引物进行进一步配对及筛选,得到最优特异性检测引物。同时,由病毒全长cDNA扩增出NP基因,琼脂糖凝胶电泳检测NP基因的扩增情况并对目的条带进行切胶回收及纯化,对回收后的NP全长基因进行核酸浓度测定,并换算成拷贝数,作为定量标准品。结果应用ABI公司的Power SYBR Green PCR MasterMix及StepOne实时荧光定量PCR仪,该检测系统灵敏度可达102 copies/μL,不同梯度标准品间线性关系（R2）达0.999,斜率为-0.3433,扩增效率为95.572%,所有标准品均在83.2℃出现尖且窄的特异性熔解峰。结论利用该检测系统可以快速定量检测季节性流感病毒H1N1,灵敏度高,可用作基础及临床实验室对季节性流感病毒H1N1感染的辅助诊断方法和临床效果的监测手段,对实验操作者要求相对较低,具有实际的应用价值。     

埃博拉病毒莱斯顿亚型实时荧光定量PCR检测方法的建立

本研究的目的为建立一种灵敏、快速、定量检测埃博拉病毒莱斯顿亚型核酸的实时荧光定量PCR检测方法及试剂盒。首先选择埃博拉病毒莱斯顿亚型的保守基因NP基因作为检测靶目标,筛选出保守序列并设计合成一对特异性引物。然后将连有NP全长基因的重组质粒作为定量标准品,将10倍系列稀释的重组质粒进行荧光定量PCR扩增,绘制标准曲线,并进行重复性、灵敏度及特异性检测。结果显示建立的埃博拉病毒莱斯顿亚型荧光定量PCR检测方法,其灵敏度可达102拷贝/μL,不同梯度标准品间线性关系(R2)达0.997,斜率为-0.3101,扩增效率为110.145%,所有标准品均在79.94℃出现尖且窄的特异性熔解峰。利用该检测系统可以快速定量检测埃博拉病毒莱斯顿亚型核酸,灵敏度高、重复性好,可用于基础及临床实验室对埃博拉病毒莱斯顿亚型感染的快速诊断和临床效果的监测,具有实际的应用价值。     

HBV感染者外周血PBMC中TLR4 mRNA与病毒载量的相关性研究

目的探讨乙型肝炎病毒感染者外周血PBMC中TLR mRNA与乙肝病毒复制的相关性。方法采用逆转录PCR检测外周血单个核细胞（PBMC）中TLR4 mRNA的含量,实时荧光定量Real Time PCR的方法检测HBV DNA,进行相关性分析。结果不同病毒载量组（〈1×103copies/μg DNA,1×103copies/μg DNA〈且〈1×105copies/μg DNA,〉1×105copies/μg DNA）TLR4 mRNA水平差异具有显著性（P〈0.01）,HBV病毒载量的对数值与TLR4 mRNA的含量存在负相关（r=-0.537,P〈0.01）。TLR4 mRNA的相对表达量与患者的ALT、AST呈正相关（r=0.608、r=0.659,P〈0.01）。结论HBV在患者体内复制活跃、病毒载量增高与外周血单个核细胞TLR4mRNA的表达下调有关。     

SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

目的 建立SYBR Green Ⅰ荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数.方法 巢式RT-PCR扩增SIV病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒.该质粒经限制性内切酶Not I酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品.结果 应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL.结论 制备的RS外标准品纯度高,SYBR Green Ⅰ荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数.     

Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.     

Low plasma human immunodeficiency virus type 2 viral load is independent of proviral load: low virus production in vivo

Levels of virus in the plasma are closely related to the pathogenicity of human immunodeficiency virus type 1 (HIV-1). HIV-2 is much less pathogenic than HIV-1, and infection with HIV-2 leads to significantly lower plasma viral load. To identify the source of this difference, we measured both viral RNA and proviral DNA in matched samples from 34 HIV-2-infected individuals. Nearly half had undetectable viral RNA loads (<100 copies/ml), but levels of proviral DNA were relatively high and confirmed that quantities of provirus in HIV-1 and HIV-2 infection were similar. Overall, HIV-2 proviral DNA load did not correlate with viral RNA load, and higher viral RNA load was associated with increased production of plasma virus from the proviral template. These results suggest that low viral load in HIV-2 infection is due to decreased rates of viral production, rather than differences in target cell infectivity.     

Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease.

Studies of cultivatable human immunodeficiency virus type 1 (HIV-1) from plasma samples from infected patients have shown a correspondence between increasing viral burden and disease progression, but these measurements are selective and thus nonrepresentative of the in vivo viral load. Quantitation of proviral DNA sequences by the polymerase chain reaction in purified CD4+ T cells has shown a similar relationship but does not provide a measure of viral gene expression. We have studied viral DNA, genomic RNA, and spliced mRNA expression of HIV-1 in infected patients with a quantitative polymerase chain reaction assay. Viral RNA expression is detected in all stages of infection. These data show that the natural history of HIV infection is associated with a shift in the balance of viral expression favoring the production of genomic RNA without a preceding period of true viral latency.     

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.     

Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.