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。高危型人乳头瘤病毒导致宫颈癌，这是全球女性癌症死亡的第二大原因，每年有28.8万人死亡。拟议工作的目标是开发一种基于dna的HPV致癌菌株诊断测试，这种测试比目前可用的测试更快，假阳性和假阴性显著降低。在I期研究中，Trevigen建立了一种名为LAMP（环介导扩增）的联合实时DNA扩增技术的原理证明，该技术由嗜热DNA修复酶（SNIPases）切割，该固定探针针对HPV16的E7基因，HPV16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HPV16靶DNA，在侧流膜上检测到彩色带。之所以选择横向流动平台，是因为它简单，而且读数不到10分钟。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，使用来自嗜气热杆菌的一种新的超耐热AP内切酶PA-Endo IV，在一个被称为PCR-SNIPase或LAMP-SNIPase的过程中，从开始到结束在1.5小时内在试纸上形成一条彩色带。DNA扩增与SNIPase反应的结合将减少假阳性和阴性，因为PA-Endo IV只有在固定探针与适当的致癌HPV毒株靶DNA完美杂交时才会切割固定探针。作为II期应用的一部分，该反应将进行多重处理，以便在4个反应中使用4个径向侧流装置检测和鉴定所有13种HPV致癌株。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验证。二期资金将用于开发一种强大而快速的检测和识别HPV致癌菌株的测试方法，这将大大减少假阳性的发生率，从而减少对阴道镜检查的需求，这是一种昂贵且侵入性的手术。该检测方法的简单性将使其能够在一种新的有效的“筛查和治疗”程序中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.