PR COMPREHENSIVE CTR FOR HIV DISPARITIES: PSM: LAB CORE

HIV 差异的公关综合点击率：PSM：实验室核心

• 批准号: 7724746
• 负责人: Yasuhiro Yamamura
• 金额: $ 32.03万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724746
• 关键词: AIDS/HIV problem; Area; Baltimore; Biohazardous Substance; Biological Assay; Blood; Blood capillaries; Budgets; CD4 Positive T Lymphocytes; Clinical; Clinical Psychology; Color; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence; DNA Sequence Analysis; Data; Detection; Development; Diagnosis; Doctor of Medicine; Doctor of Philosophy; Educational workshop; Enrollment; Ensure; Enzyme-Linked Immunosorbent Assay; Evaluation; FOLH1 gene; Faculty; Fostering; Funding; Future; Gagging; Gene Expression; Genetic; Genotype; Grant; HIV; HIV diagnosis; HIV drug resistance; Heating; Hepatitis C virus; Housing; Human Papillomavirus; Human Virology; Individual; Institutes; Institution; Laboratories; Maryland; Mental Health; Methods; Monitor; Mutation Analysis; National Institute of Mental Health; Nature; Numbers; Patients; Phase; Plasma; Polymerase Chain Reaction; Preparation; Procedures; Production; Progress Reports; Proteins; Puerto Rico; Reaction; Reporting; Research; Research Infrastructure; Research Personnel; Research Project Grants; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Running; Sampling; Series; Services; Source; Staging; Structure; System; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Time; Training; United States National Institutes of Health; Universities; Update; Validation; Variant; Viral Load result; Western Blotting; base; capillary; chemokine; cost; cytokine; day; immunological status; instrument; programs; prospective; research clinical testing; AIDS/HIV problem; Area; Baltimore; Biohazardous Substance; Biological Assay; Blood; Blood capillaries; Budgets; CD4 Positive T Lymphocytes; Clinical; Clinical Psychology; Color; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence; DNA Sequence Analysis; Data; Detection; Development; Diagnosis; Doctor of Medicine; Doctor of Philosophy; Educational workshop; Enrollment; Ensure; Enzyme-Linked Immunosorbent Assay; Evaluation; FOLH1 gene; Faculty; Fostering; Funding; Future; Gagging; Gene Expression; Genetic; Genotype; Grant; HIV; HIV diagnosis; HIV drug resistance; Heating; Hepatitis C virus; Housing; Human Papillomavirus; Human Virology; Individual; Institutes; Institution; Laboratories; Maryland; Mental Health; Methods; Monitor; Mutation Analysis; National Institute of Mental Health; Nature; Numbers; Patients; Phase; Plasma; Polymerase Chain Reaction; Preparation; Procedures; Production; Progress Reports; Proteins; Puerto Rico; Reaction; Reporting; Research; Research Infrastructure; Research Personnel; Research Project Grants; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Running; Sampling; Series; Services; Source; Staging; Structure; System; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Time; Training; United States National Institutes of Health; Universities; Update; Validation; Variant; Viral Load result; Western Blotting; base; capillary; chemokine; cost; cytokine; day; immunological status; instrument; programs; prospective; research clinical testing

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Laboratory Core The specific aims of the core laboratory activity remain unchanged except that some additional tests are now available. The laboratory core will provide HIV related testing for clinical evaluation of patients enrolled in one or the other ongoing as well as developing projects under the auspice of the Puerto Rico CCHD-HIV. The following tests are implemented through the core. 1. HIV and HCV ELISA and HIV Western blot analyses. 2. Flow cytometric analysis of CD4 T cell enumeration, intracytoplastic cytokine production by T cell subsets and quantification of extra-cellularly produced cytokines-chemokines. 3. HIV and HCV viral load assessment by Roche Amplicor procedures. 4. HCV Genotyping by the line-probe assay (LIPA). 5. HIV drug resistance mutation analysis (genotyping) by the Bayer (Visible Genetics) TruGene procedure. 6. Flow cytometric analysis of HIV infected T cells. 7. HIV clade (genotype) analysis by DNA sequencing. 8. Human papillomavirus (HPV) genotyping methods have been incorporated into the core service. Progress report (1) The nature of the HIV and HCV diagnosis tests remain the same as reported previously reported: namely, no new diagnosis of HIV or HCV was performed during this period, and all core tests provided were for characterization and staging of HIV infected individuals who had been diagnosed prior to enrollment in the CCHD projects. (2) Updating of HIV and HCV Testing Formats. 2a) During the last project year, Roche HCV test format was switched from the "Amplicor" PCR-ELISA to the "TaqMan" real-time RT-PCR. Amplicor HIV Monitor test format was also switched during the current year period to TaqMan real-time PCR. Both test systems are equipped with Ampli-Prep automated sample preparation workstation, which reduces technical variations as well as the biohazard risk to operators. Updating of technologists on these procedures were also completed during this period 2b)Homebrew procedure for HIV viral load assessment by real-time RT-PCR (HIV gag-probe specific) has been validated. A partial validation data are shown in Figure 1. This validated procedure may be utilized, when a much larger than budgeted amount of tests is required for the projects. It should be considered a viable, less costly alternative to the FDA-approved kit-based procedures that have been offered through the core. 2c)HIV genotyping based on a homebrew DNA sequencing using Visible-Genetics long Run Tower automated DNA sequencers has been validated. This procedure shall significantly reduce the costs of HIV genotyping from the current ca. $250 to $100 or lower. A new, high capacity automated DNA sequencer, ABI 3730 with 48 capillaries was purchased by the PSM for the RCMI and CCHD activities. When fully validated (anticipated in August 2007), it will be possible to complete DNA sequencing in a much shorter time and the costs will also be significantly reduced. For example, HIV genotyping may be provided, if requested, at below $100 for CCHD investigators. 2d)Layout of the DNA/RNA sample preparation room for clinical PCR such as Ampli-Taq and COBAS (Roche) was significantly modified to accommodate the newly implemented the Roche test format change from Amplicor to AmpliTaq. In addition, separate rooms were assigned for template-free room for mastermix preparation, PCR amplification, and DNA sequencing. The last room currently houses 3 VG Long-Run Tower DNA Sequencers and one ABI 3730 48 capillaries DNA Sequencer. Additional cooling units were installed in the DNA sequencing room to accommodate the extra heat generated by the new instrument. Separation of each room for separate procedure minimizes the potential risk of template carry over (contamination) from one room to the other. Laboratory coats for each laboratory are identified by 3 different colors to ensure that no laboratory coat will be moved to another room. Future plans: (1) During the 2007 annual CCHD investigators' retreat in Ponce, certain new developments in expansion of ongoing research projects necessitated the core service of cytokine-chemokine quantification in the blood of patients undergoing evaluation. Assessments of intra-cellular and extra-cellular cytokines/chemokines (including those in the patients' blood/plasma) were requested by investigators for better assessments of individuals' mental health and immunological status. Quantification of cytokines (chemokines) or their gene expression by protein- or genetic-arrays will be standardized during this period to assist further development of such research projects through the CCHD initiative. (2)In order to promote the development of research projects in AIDS/HIV associated mental health areas, a workshop has been organized to facilitate the research infrastructure/project development using the existing Clinical Psychology program of the PSM. The workshop will be held on June 19-20, 2007, at the PSM with the following outside panelists: C. Zorrilla, M.D. (UPR, CCHD PI), S. Loue, Ph.D., J.D. (Case Western Reserve University, Cleveland, OH), L. Temoshok, Ph.D.(Institute of Human Virology, U-Maryland at Baltimore, MD), and D. Stoff, Ph.D. (NIMH). The workshop will, in addition to fostering the development of research projects by junior investigators, develop the mental health core infrastructure which may further contribute to the CCHD activities. The 2 day workshop may help identify potential research projects that may be included in the next phase CCHD structure. (3)It is planned that during the neat program year period, a series of meetings will be organized with the current and prospective CCHD faculties for identifying the emerging needs for the Laboratory Core function. As described above, as projects progress, different technical needs will be identified. Since a new competitive renewal proposal of the CCHD program will begin to be organized during the next year, and a number of projects or project areas that will be included in the next phase program will be identified, the infrastructure of the Laboratory Core also requires adjustments. New technical developments and training of technologists to accommodate the needs will be implemented shortly thereafter. For example, as described above (1), a microarray detection system using chimoluminescence reaction has already been installed in the laboratory. Pilot testings of cytokine-chemokine microarrays are being standardized. The tests may be available shortly after the summer of 2007.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 实验室核心 核心实验室活动的具体目的保持不变，除了现在可以进行一些其他测试。 实验室核心将提供与HIV相关的测试，以对在波多黎各Rico CCHD-HIV的主持下进行的一个或另一个正在进行的患者进行临床评估。 以下测试通过核心实现。 1。HIV和HCV ELISA和HIV Western印迹分析。 2。对CD4 T细胞枚举，T细胞亚群产生的细胞因子产生的流式细胞仪分析以及细胞外产生的细胞因子化学因子的定量。 3。通过Roche扩增器程序评估HIV和HCV病毒载荷。 4。线索探针测定法（LIPA）的HCV基因分型。 5。拜耳（可见遗传学）Trugene程序的HIV耐药性突变分析（基因分型）。 6。对HIV感染的T细胞的流式细胞仪分析。 7。通过DNA测序分析HIV进化枝（基因型）。 8。人乳头瘤病毒（HPV）基因分型方法已纳入核心服务。 进度报告 （1）HIV和HCV诊断测试的性质与先前报道的：即在此期间没有进行HIV或HCV的新诊断，并且提供的所有核心测试均用于对CCHD项目入学前已诊断出的HIV感染者的表征和分期。 （2）更新HIV和HCV测试格式。 2A）在上一年，Roche HCV测试格式从“放大器” PCR-Elisa转换为“ Taqman”实时RT-PCR。在本年期，还将放大器HIV监测器测试格式转换为Taqman实时PCR。 两种测试系统都配备了Ampli-PREP自动样品准备工作站，从而降低了技术变化以及对操作员的生物危害风险。在此期间，还完成了有关这些程序的技术人员的更新 2B）通过实时RT-PCR（HIV GAG-PROBE特异性）进行HIV病毒负荷评估的自制程序已验证。部分验证数据如图1所示。当项目需要大于预算的测试时，可以利用此验证的过程。它应该被认为是通过核心提供的基于FDA批准的套件程序的可行，成本较低的替代方法。 2C）使用可见的基因测序基于自制的DNA测序的HIV基因分型已验证。 该程序应大大降低目前CA的HIV基因分型成本。 $ 250至$ 100或更低。 PSM购买了一种新的，高容量的自动化DNA测序仪，ABI 3730，带有48个毛细血管，用于RCMI和CCHD活动。如果经过全面验证（预计于2007年8月），则可以在短时间内完成DNA测序，并且成本也将大大降低。 例如，如果需要，可以为CCHD调查人员提供低于100美元的HIV基因分型。 2d）为临床PCR（例如Ampli-TAQ和Cobas（Roche））的DNA/RNA样品准备室的布局进行了显着修饰，以适应新实施的Roche测试格式从扩展器到Amplitaq的变化。 此外，分配了单独的房间，以用于无模板的房间，用于策划策划，PCR扩增和DNA测序。 最后一个房间目前装有3个VG长期塔DNA测序仪和一个ABI 3730 48毛细管DNA Sequencer。在DNA测序室中安装了其他冷却单元，以容纳新乐器产生的额外热量。 每个房间进行单独过程的分离可以最大程度地减少模板的潜在风险（污染）从一个房间到另一个房间。每个实验室的实验室大衣都用3种不同的颜色确定，以确保不会将实验室外套移至另一个房间。 未来计划： （1）在2007年年度CCHD调查人员在Ponce的撤退期间，正在进行的研究项目的某些新发展需要在接受评估的患者血液中为细胞因子化学化的量化核心服务。研究人员要求评估细胞内和细胞外细胞因子/趋化因子（包括患者血液/血浆中的因子），以更好地评估个体的心理健康和免疫学状况。 在此期间，将标准化细胞因子（趋化因子）或其基因表达，以通过CCHD计划帮助进一步开发此类研究项目。 （2）为了促进艾滋病/艾滋病毒相关心理健康领域的研究项目的发展，已经组织了一个研讨会，以利用PSM的现有临床心理学计划来促进研究基础设施/项目开发。研讨会将于2007年6月19日至20日在PSM举行，以下外部小组成员：C。Zorrilla，M.D。（UPR，UPR，CCHD PI），S。Loue，J.D。 （nimh）。 该研讨会除了促进初级研究人员的研究项目发展外，还将开发精神健康核心基础设施，这可能进一步有助于CCHD活动。为期2天的研讨会可能有助于确定可能包括在下一阶段CCHD结构中的潜在研究项目。 （3）计划在整洁的计划期间，将与当前和潜在的CCHD学院组织一系列会议，以确定实验室核心功能的新兴需求。如上所述，随着项目的进展，将确定不同的技术需求。由于将在明年开始组织CCHD计划的新竞争续约建议，并且将确定将包括许多项目或项目领域的项目或项目领域，因此实验室核心的基础设施也需要调整。此后不久将实施新的技术发展和培训以适应需求。例如，如上所述（1），实验室已经安装了使用嵌合发光反应的微阵列检测系统。 细胞因子化学微阵列的试点测试正在标准化。 这些测试可能会在2007年夏后不久提供。