SOLID PHASE PLATE ASSAY (SPPA) FOR HIV1 GENOTYPING

建立了一种基于核酸的固相微孔板捕捉法，用于区分M和O群的主要HIV-1亚型。样品处理条件进行了优化，以确保该方法的灵敏度和重复性。在提取、逆转录、聚合酶链式反应扩增和检测过程中，设计了含有合成片段和相应序列的内控载体，用于监测样品的回收。从HIV-1 env保守区选择生物素标记的简并引物，以使所有已知的HIV-1分离株，包括O型，都能被扩增。通过与代表每个指定亚型的特定固定化捕获低聚物杂交，将包含env高变区的标记RT-PCR扩增片段捕获在单独的微滴定井上，并通过使用平板阅读器的显色反应进行比色检测。对从不同地区收集的数十个HIV-1病毒分离株进行了测试，并通过对病毒Gag和env区域的直接测序确认了亚型指定。这种新发展的微板捕捉法可以检测和区分所有主要的HIV-1亚型，包括A、B、C、D、E、F和HIV-1 O亚型。这种基因分型方法可以作为一种敏感和可靠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：该检测方法将提供一种对临床样本进行艾滋病毒-L分型的方法，并用于生产可用于候选疫苗和抗病毒药物开发的分子对照和试剂盒，供医生评估艾滋病毒-L毒株的抗逆转录病毒治疗和耐药性，以及供科学家和临床实验室评估艾滋病毒-L检测方法的性能。微型设备、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格式，以创建艾滋病毒-L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.