SOLID PHASE PLATE ASSAY (SPPA) FOR HIV1 GENOTYPING

用于 HIV1 基因分型的固相板测定 (SPPA)

• 批准号: 6170782
• 负责人: JIUPING J JI
• 金额: $ 31.7万
• 依托单位: SERACARE BIOSERVICES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170782
• 关键词: genetic strain; genetic techniques; genotype; human immunodeficiency virus 1; nucleic acid hybridization; polymerase chain reaction; solid state; technology /technique development; virus genetics

A nucleic acid based solid phase microplate capture assay is being developed for the differentiation of the major HIV-1 subtypes of groups M and O. Sample processing conditions is optimized to ensure the assay's sensitivity and reproducibility. Ah internal control vector containing a synthetic fragment flanked by sequences corresponding to the HIV primers are designed to monitor sample recovery during extraction, reverse transcription, PCR amplification and detection. Biotin- labeled degenerate primers from the conserved region of HIV-1 env are selected such that all known HIV-1 isolates, including type O, could be amplified. The labeled RT-PCR amplicons containing the hyper-variable region of env are captured on separate microtiter wells by hybridization to specific immobilized capture oligomers representing each of the designated subtypes, and colorimetrically detected by a chromogenic reaction using a plate reader. Several dozen of HIV- 1 viral isolates collected from different geographic regions are tested and subtype designations are confirmed by direct sequencing of viral gag and env regions. This newly developed microplate capture assay can detect and differentiate all major subtypes of HIV-1, including subtypes A, B, C, D, E, F and HIV-1 subtype O. This genotyping assay can be used as a sensitive and robust screening and genotyping method for monitoring the extent of global HIV-1 variation and for disease management. PROPOSED COMMERCIAL APPLICATION: The assay will provide a method for HIV-l subtyping of clinical samples and for the production of molecular controls and kits useful for developers of candidate vaccines and antivirals, to physicians in the assessment of anti- retroviral treatment and drug resistance of HIV-l strains, and to scientists and clinical laboratories to evaluate the performance of HIV-l detection assays. Manufacturers of microdevices, microchips and automated systems could adapt this assay into a biochip format to create a HIV-l genotyping system.

正在开发一种基于核酸的固相微孔板捕获测定法，用于区分 M 组和 O 组的主要 HIV-1 亚型。优化样品处理条件以确保测定法的灵敏度和重现性。内部对照载体含有侧翼对应于 HIV 引物的序列的合成片段，旨在监测提取、逆转录、PCR 扩增和检测过程中的样品回收率。选择来自 HIV-1 env 保守区域的生物素标记的简并引物，以便可以扩增所有已知的 HIV-1 分离株，包括 O 型。含有 env 高变区的标记 RT-PCR 扩增子通过与代表每种指定亚型的特定固定化捕获寡聚物杂交而捕获在单独的微量滴定孔上，并使用读板器通过显色反应进行比色检测。对从不同地理区域收集的数十种 HIV-1 病毒分离株进行了测试，并通过病毒 gag 和 env 区域的直接测序来确认亚型名称。这种新开发的微孔板捕获测定可以检测和区分 HIV-1 的所有主要亚型，包括 A、B、C、D、E、F 亚型和 HIV-1 O 亚型。这种基因分型测定可用作敏感且稳健的筛查和基因分型方法，用于监测全球 HIV-1 变异的程度和疾病管理。拟议的商业应用：该测定将为临床样本的 HIV-1 亚型分型以及分子对照和试剂盒的生产提供一种方法，这些对照和试剂盒可用于候选疫苗和抗病毒药物的开发人员、医生评估抗逆转录病毒治疗和 HIV-1 毒株的耐药性以及科学家和临床实验室评估 HIV-1 检测测定的性能。微型设备、微芯片和自动化系统的制造商可以将该测定法改编成生物芯片格式，以创建 HIV-1 基因分型系统。