SOLID PHASE PLATE ASSAY (SPPA) FOR HIV1 GENOTYPING

目前正在开发一种基于核酸的固相微孔板捕获法，用于区分HIV-1主要亚型M组和o组。样品处理条件经过优化，以确保检测的敏感性和重复性。Ah内控载体含有与HIV引物相对应的合成片段，用于在提取、反转录、PCR扩增和检测过程中监测样品回收率。从HIV-1环境的保守区域选择生物素标记的简并引物，这样所有已知的HIV-1分离株，包括O型，都可以扩增。标记的RT-PCR扩增子包含env的高可变区，通过与代表每个指定亚型的特定固定捕获低聚物杂交，在单独的微滴孔上捕获，并使用板阅读器通过显色反应进行比色检测。对从不同地理区域收集的几十株HIV- 1病毒分离株进行了检测，并通过对病毒gag区和env区进行直接测序来确定亚型。这种新开发的微孔板捕获试验可以检测和区分HIV-1的所有主要亚型，包括亚型A、B、C、D、E、F和HIV-1亚型o。这种基因分型试验可以作为一种敏感和强大的筛选和基因分型方法，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：该检测将为临床样本的hiv - 1亚型分型提供一种方法，并为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菌株的耐药性以及科学家和临床实验室评估hiv - 1检测检测方法的性能提供一种分子控制和试剂盒。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编成生物芯片格式，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.