SOLID PHASE PLATE ASSAY (SPPA) FOR HIV1 GENOTYPING

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的灵敏度和重现性。设计了含有合成片段的内部对照载体，该合成片段侧翼为对应于HIV引物的序列，用于监测提取、逆转录、PCR扩增和检测期间的样品回收率。选择来自HIV-1 env保守区的生物素标记的简并引物，使得所有已知的HIV-1分离株（包括O型）都可以扩增。通过与代表每种指定亚型的特异性固定捕获寡聚体杂交，将含有env高变区的标记RT-PCR扩增子捕获在单独的微量滴定威尔斯孔上，并使用酶标仪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病毒分离株进行检测，并通过病毒gag和env区域的直接测序确认亚型名称。这种新开发的微孔板捕获检测方法可以检测和区分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。这种基因分型检测可用作一种灵敏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。拟定商业应用：该测定将提供用于临床样品的HIV-I亚型分型和用于生产分子对照和试剂盒的方法，所述分子对照和试剂盒可用于候选疫苗和抗病毒药的开发者、可用于医生评估抗逆转录病毒治疗和HIV-I菌株的耐药性、以及可用于科学家和临床实验室评估HIV-I检测测定的性能。微设备、微芯片和自动化系统的制造商可以将这种检测方法改造成生物芯片形式，以创建HIV-1基因分型系统。

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.