Herpes virus typing assay for detecting genital herpes

描述（由申请人提供）：我们的NIAID生物防御I期SBIR计划的目标是开发和商业化用于生物防御应用的现场可部署核酸分析设备。我们已经开始销售Isoacetylase II试剂盒，这将使我们的客户能够开发自制检测试剂（我们在商业化计划中列出了客户）。例如，LANL已获得NIH资助（R 01），以开发使用我们的Isoacetamide II测定制剂检测流感病毒的测定系统（http：//www.lanl.gov/science/1663/flu.php）。这些试剂盒含有在我们的I期中发现的增强子蛋白，其加速解旋酶依赖性扩增（HDA）;即，一种热稳定单链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）的特异性扩增。此外，我们优化了引物设计过程，以促进检测开发。我们还与Keck Graduate Institute（KGI）合作开发了一种小型便携式核酸分析设备（又名Rabbit）。最后，我们已经开发了许多基于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BioHessel建立一个有效的QMS，生产cGMP试剂和执行我们检测的设备，并验证我们的检测系统，用于大量（约1，000）临床样本的HSV-1和HSV-2分型。本临床研究生成的数据将用于申请FDA对本诊断系统销售的监管许可。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，HSV DNA的分子检测比传统的病毒培养具有更高的灵敏度（灵敏度超过95%，而培养的灵敏度为75%）。尽管如此，由于分子检测的成本较高，因此分子检测并不像培养那样广泛用于生殖器疱疹的诊断。此外，分子检测被视为一个复杂的过程，需要从临床样本中分离DNA。最后，由于没有FDA批准的用于HSV的商业分子测定，在临床实验室中实施这样的测试是繁重的。我们相信我们可以为这个问题提供一个解决方案。在Cellmatics Transport Kit缓冲液（Becton Dickinson，Sparks，MD）中采集的生殖器和口腔拭子样本可稀释1/2000，用于通过Isocyanine HSV检测试剂盒进行直接分析。这种基于稀释的样品处理将大大降低我们的分析系统的复杂程度。此外，我们已经开始与GE Healthcare合作，为我们的检测开发Ready-To-Go（tm）Isoclonal配方，这将消除对多次精确体积测量的需求。BioHestival和GE Healthcare计划在设计控制下开发一种制剂，以便根据cGMP进行生产。KGI开发的仪器也将转移到cGMP制造商（Source Scientific），因此，到2009年年中，我们预计将有一个cGMP检测系统准备用于临床验证研究。

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.