Herpes virus typing assay for detecting genital herpes

描述(由申请人提供)：我们NIAID生物防御第一阶段SBIR计划的目标是开发用于生物防御应用的现场可部署核酸分析设备，并将其商业化。我们已经开始销售IsoAmp II试剂盒，这将允许我们的客户开发家庭酿造测试(我们将客户列入商业化计划)。例如，LANL已经获得了美国国立卫生研究院的资助(R01)，以开发一种使用我们的IsoAmp II检测配方(http://www.lanl.gov/science/1663/flu.php).)来检测流感病毒的检测系统这些试剂盒包含一种增强子蛋白，在我们的第一阶段中发现，它可以加速解旋酶依赖的扩增(HDA)；即，一种耐热的单链DNA结合蛋白。HDA化学现在允许核糖核酸(RNA)和脱氧核糖核酸(DNA)的特定扩增。此外，我们还优化了我们的引物设计流程，以促进检测方法的开发。我们还与凯克研究所(KGI)合作开发了一种小型便携式核酸分析设备(又名。兔子)。最后，我们开发了一些基于DNA探针的生物防御靶标检测方法，例如炭疽杆菌和埃博拉病毒。在第二阶段，我们建议在BioHelix建立一个有效的QMS，生产执行我们检测的cGMP试剂和设备，并用大量(约1,000)个临床样本验证我们的检测系统用于HSV-1和HSV-2的分型。这项临床研究产生的数据将用于向FDA申请销售该诊断系统的监管许可。我们选择HSV作为第一次临床检测的目标，因为这种病毒是美国性传播疾病(STD)的主要原因。此外，单纯疱疹病毒DNA的分子检测比传统的病毒培养具有更高的敏感性(敏感性超过95%，而培养的敏感性为75%)。尽管如此，由于分子检测的成本较高，分子检测在生殖器疱疹诊断中的应用不如培养法广泛。此外，分子检测被认为是一个复杂的过程，需要从临床样本中分离出DNA。最后，由于没有FDA批准的针对HSV的商业分子检测，在临床实验室实施这样的检测是繁重的。我们相信我们可以为这个问题提供解决方案。收集在Cellmatics Transport Kit Buffer(Becton Dickinson，Sparks，MD)中的生殖器和口腔拭子样本可以稀释1/2000，用于IsoAmp HSV直接分析。这种基于稀释的样品处理将极大地降低我们检测系统的复杂程度。此外，我们已经开始与GE Healthcare合作，为我们的检测开发一种即用型(Tm)IsoAmp配方，该配方将不再需要多次精确的体积测量。Biohelix和GE Healthcare计划开发一种设计控制下的配方，以便在cGMP下生产。KGI开发的仪器还将转移到cGMP制造商(来源科学公司)，这样到2009年年中，我们预计将有一个cGMP分析系统准备好用于临床验证研究。

DESCRIPTION (provided by applicant): The objective of our NIAID Biodefense Phase I SBIR program was to develop, and commercialize field deployable nucleic acid analysis devices for use in biodefense applications. We have begun to sell IsoAmp II kits that will allow our clients to develop home brew assays (we list customers in the commercialization plan). For example, the LANL has obtained NIH funding (R01) to develop an assay system for detecting influenza virus using our IsoAmp II assay formulation (http://www.lanl.gov/science/1663/flu.php). These kits contain an enhancer protein, discovered in our Phase I, that accelerates helicase dependent amplification (HDA); i.e., a thermostable single strand DNA binding protein. HDA chemistry now allows for the specific amplification of both ribonucleic acids (RNA), and deoxyribonucleic acids (DNA). Moreover, we have optimized our primer design process to facilitate assay development. We also collaborated with the Keck Graduate Institute (KGI) to develop a small, portable nucleic acid analysis device (a.k.a. Rabbit). Finally, we have developed a number of DNA probe based assays for biodefense targets; e.g. Bacillus anthracis and Ebola virus. In this Phase II, we propose to put into place a functioning QMS at BioHelix, manufacture cGMP reagents and devices that perform the our assays, and to validate our assay system for typing HSV-1 & HSV-2 with a large number (~1,000) clinical samples. The data generated from this clinical study will be used to apply for regulatory clearance from FDA for the sale of this diagnostic system. We have selected HSV as a target for this first clinical assay because this virus is the lead cause of sexually transmitted disease (STD) in the United States. In addition, molecular testing for HSV DNA has greater sensitivity than the traditional viral culture (sensitivity of more than 95 percent, compared with 75 percent for culture). Despite this fact, molecular tests are not as widely used as culture for the diagnosis of genital herpes because of the higher cost of molecular tests. Moreover, molecular testing is viewed as a complex process requiring the isolation of DNA from clinical samples. Finally, as there are no FDA cleared commercial molecular assays for HSV, implementing such tests in clinical laboratories is burdensome. We believe we can offer a solution to this problem. Genital and oral swab specimens collected in Cellmatics Transport Kit buffer (Becton Dickinson, Sparks, MD) can be diluted 1/2000 for direct analysis by the IsoAmp HSV assay. This dilution based sample processing will greatly reduce the degree of complexity of our assay system. In addition, we have begun to collaborate with GE Healthcare to develop a Ready-To-Go(tm) IsoAmp formulation for our assays that will eliminate the need for multiple, precise volumetric measurements. BioHelix and GE Healthcare plan to develop a formulation under design control in order to manufacture it under cGMP. The instrument developed by KGI will also be transferred to a cGMP manufacturer (Source Scientific), such that by mid-2009 we expect to have a cGMP assay system ready for clinical validation studies.