DNA-Based Lateral Flow Assay for Oncogenic Strains of HPV

描述（由申请人提供）：人乳头瘤病毒（HPV）包括一个超过100种不同毒株的病毒家族。HPV的高风险菌株导致宫颈癌，这是全球女性癌症死亡率的第二大原因，每年有288，000人死亡。拟议工作的目标是开发一种基于DNA的HPV致癌株诊断测试，该测试比目前可用的测试更快速，假阳性和假阴性率显着降低。在第一阶段，Trevigen建立了一种名为LAMP（环介导扩增）的组合实时DNA扩增技术的主要证据，该技术通过嗜热DNA修复酶（SNIPases）切割固定化探针，靶向HPV 16的E7基因，HPV 16是一种致癌菌株，导致超过50%的宫颈癌。切割产物的存在，诊断HPV 16靶DNA，检测为侧流膜上的有色条带。选择侧流平台是因为其简单性和在不到10分钟内读出。在第二阶段的资助下，我们建议优化实时DNA扩增（PCR或LAMP）和SNIPase切割反应，使用来自嗜气热芽孢杆菌的一种新的超嗜热AP内切核酸酶PA-Endo IV，在一个称为PCR-SNIPase或LAMP-SNIPase的过程中，从开始到结束在1.5小时内在试纸上产生一条彩色条带。DNA扩增与SNIPase反应的组合将减少假阳性和假阴性，因为PA-Endo IV仅在固定化探针与其适当的致癌HPV株靶DNA完美杂交时才切割固定化探针。该反应将被多重化，以允许使用四个径向侧向流装置在四个反应中检测和鉴定HPV的所有13种致癌菌株，该装置将作为该II期申请的一部分开发。最后，我们优化的HPV检测将在一组高危患者的存档宫颈样本上进行测试和鉴定。第二阶段的资金将导致开发一种用于检测和鉴定HPV致癌株的强大而快速的测试，这将大大降低假阳性的发生率，从而减少对阴道镜检查的需求，这是一种昂贵的侵入性程序。该检测方法的简单性将使其在一个新的有效的“筛选和治疗”程序中实施，用于服务不足的人群。

DESCRIPTION (provided by applicant): Human Papilloma Virus (HPV) comprises a family of viruses of over 100 different strains. The high-risk strains of HPV cause cervical cancer, which is the second leading cause of female cancer mortality worldwide with 288,000 deaths yearly. The goal of the proposed work is to develop a DNA-based diagnostic test for the oncogenic strains of HPV that is more rapid and has significantly lower false positives and false negatives than the currently available test. In Phase I, Trevigen established proof-of -principal for a combined real-time DNA amplifacation technique called LAMP (loop-mediated amplification) with cleavage by thermophilic DNA repair enzymes (SNIPases), of an immobilized probe targeted to the E7 gene of HPV16, an oncogenic strain that causes over 50% of all cervical cancers. The presence of the cleavage product, diagnostic for the HPV16 target DNA, was detected as a colored band on a lateral flow membrane. The lateral flow platform was chosen because of its simplicity and readout in less than 10 minutes. Under Phase II funding, we propose to optimize the real-time DNA amplification (PCR or LAMP) and the SNIPase cleavage reaction with a new hyperthermophilic AP endonuclease from Pyrobaculum aerophilum called PA-Endo IV, in a process known as PCR-SNIPase or LAMP-SNIPase, leading to a colored band on a dipstick within 1.5 hour from start to finish. The combination of the DNA amplification with the SNIPase reaction will reduce false positives and negatives because PA-Endo IV only cleaves the immobilized probes only when they are perfectly hybridized to their appropriate oncogenic HPV strain target DNA. The reaction will be multiplexed to allow detection and identification of all 13 oncogenic strains of HPV in four reactions using four radial lateral flow devices to be developed as part of this Phase II application. Finally, our optimized HPV test will be tested and qualified on archived cervical samples from a cohort of high-risk patients. The Phase II funding will lead to the development of a robust and rapid test for detection and identification of oncogenic strains of HPV that will significantly reduce the incidence of false positives and therefore reduce the need for colpsocopy, an expensive and invasive procedure. The simplicity of the assay will allow its implementation in a new efficacious "screen-and-treat" procedure for underserved populations.