Lateral Flow Assay for Oncogenic Strains of HPV

描述(由申请人提供)： Trevigen/TreviMed开发了错配识别DNA分析系统(MIDAS)，该系统依靠SNIPase(遗传优化的DNA错配修复酶)检测病原体DNA，而不需要PCR扩增。本第一阶段提案中概述的研究目的是使Midas适应侧向流动的油尺平台。为此，我们提出了以下具体目标：(1)设计并测试固定化MIDAS探针，用于高危人乳头瘤病毒株(HPV 16和18株)的生物素和各种半抗原，如二硝基苯基(DNP)、地高辛(DIG)、异硫氰酸荧光素(FITC)或溴脱氧尿苷(BrdU)。在HPV靶DNA和SNIPase存在的情况下，探针发生裂解，导致在侧流试纸上形成一条有色带。MIDAS探针最初将以单链寡核苷酸为目标进行优化，对应于HPV-16和HPV-18DNA序列；(2)优化Diptick-MIDAS，用于检测克隆的HPV-16和HPV-18DNA，以及来自HPV-16和HPV-18阳性细胞系的基因组DNA。这些努力将最终实现对导致宫颈癌的HPV致癌菌株的快速、敏感、多重、成本效益和准确的检测。

DESCRIPTION (provided by applicant): Trevigen/TreviMed has developed the Mismatch Identification DNA Analysis System (MIDAS) that relies on SNIPase (genetically-optimized DNA mismatch repair enzymes) for the detection of pathogen DNA without the need for PCR amplification. The purpose of the research outlined in this Phase I proposal is to adapt MIDAS to a lateral-flow dipstick platform. To this end, we propose the following specific aims: (1) to design and test immobilized MIDAS probes to high-risk oncogenic strains of human papilloma virus (HPV strains 16 and 18) that incorporate biotin and various haptens such as dinitrophenyl (DNP), digoxigenin (DIG), fluorescein isothiocyanate (FITC), or bromodeoxyuridine (BrdU). In the presence of HPV target DNA and SNIPase, cleavage of the probe occurs, leading to the formation of a colored band on a lateral-flow dipstick. The MIDAS probes will be optimized initially with single-stranded oligonucleotides as targets, corresponding to HPV-16 and HPV-18 DNA sequences; (2) to optimize Dipstick-MIDAS for detecting the presence of cloned HPV-16 and HPV-18 DNA, and genomic DNA derived from HPV-16 and HPV-18-positive cell lines. These efforts will culminate in a rapid, sensitive, multiplexed, cost-effective, and accurate assay for oncogenic strains of HPV that cause cervical cancer.