Real-time PCR Diagnostic Kit for Giardia and Cryptosporidium

描述(申请人提供)：这份为期两年的提案寻求小型企业支持IDX Labs LLC继续与弗吉尼亚大学的Houpt博士合作，将贾第鞭毛虫和隐孢子虫的敏感实时聚合酶链式反应(QPCR)检测带到美国实验室。根据疾控中心的数据，这些寄生虫每年感染多达200万美国人，我们已经开发出针对这两种感染的可靠、敏感的qPCR测试，并已在孟加拉国和坦桑尼亚的数百名患者身上成功使用。在第一阶段，这些分析被转换为隐孢子虫/贾第虫多重分析，该方法检测所有已知的人类亚种，并可在两个主要的qPCR平台(罗氏LightCycler和BioRad iCycler)上使用。在第一阶段，我们还确定了市场上可用的试剂盒中最敏感的DNA提取程序，然后通过磁性靶标DNA捕获进一步提高了PCR检测的灵敏度。这一第二阶段的应用程序现在将最终完成从粪便中提取目标DNA的程序，以实现最高的吞吐量，并从我们坦桑尼亚站点的现场收集验证数据，以便准备FDA 501(K)应用程序，我们预计这将允许强劲的市场进入。我们假设我们的技术将比典型的金标梅毒隐孢子虫/贾第虫IFA更敏感，我们将利用第三次决胜局的PCR测试来解决任何不一致的结果。其次，鉴于市场和需要改进饮用水和娱乐用水样本中的原虫测试，该测试还将在水样上与环保局的贾第鞭毛虫/隐孢子虫方法#1623进行验证。这一应用程序的创新之处在于DNA捕获提取技术，这是一个可翻译到粪便以外的其他临床标本的平台，以及将最先进的诊断方法带入抗原检测之外的概念，以满足对水传播健康重要性的资金不足的寄生虫感染。 这一第二阶段的应用将推动我们开发贾第虫/隐孢子虫实时聚合酶链式反应试剂盒，该试剂盒将高度敏感的目标特定DNA提取与高度优化的多重定量聚合酶链式反应分析相结合。在第二阶段，最终的样品到结果试剂盒将完成，并将对照现有的黄金标准IFA对临床样品进行性能验证，以使501(K)提交给FDA。

DESCRIPTION (provided by applicant): This two-year proposal seeks small business support for IDX Labs LLC to continue collaborating with Dr. Houpt at the University of Virginia to bring sensitive real-time PCR (qPCR) tests for Giardia and Cryptosporidium to U.S. labs. According to the CDC, these parasites infect up to 2 million Americans annually, and we have developed robust sensitive qPCR tests for both infections that have been used successfully on hundreds of patients in Bangladesh and Tanzania. During Phase I these assays were converted into a Cryptosporidium/Giardia multiplex assay that detected all known human subspecies and was workable on two major qPCR platforms (Roche LightCycler and BioRad iCycler). During Phase I we also determined the most sensitive DNA extraction procedure among the commercially available kits, and then improved the sensitivity of PCR detection further through magnetic target-specific DNA capture. This Phase II application will now finalize the target DNA extraction procedure from stool for highest throughput and collect validation data from the field at our site in Tanzania in order to prepare an FDA 501(k) application, which we predict will allow a strong market entry. We hypothesize that our technique will be more sensitive than the typical gold standard Merifluor Cryptosporidium/Giardia IFA, and we will utilize a third tiebreaker PCR test to resolve any discrepant results. Secondarily, the test will also be validated on water samples versus the EPA's Giardia/Cryptosporidium method #1623, given the market and need for improved tests for these protozoa on drinking and recreational water samples. The innovation of this application lies in the DNA capture extraction technology, a platform translatable to other clinical specimens besides stool, and the concept of bringing state-of-the-art diagnostics beyond antigen detection to under-funded parasitic infections of waterborne health importance. This Phase II application will advance our development of a Giardia/Cryptosporidium real time PCR kit that incorporates highly sensitive target-specific DNA extraction with a highly optimized multiplex qPCR assay. During Phase II the final sample-to-result kit will be completed and performance will be validated on clinical samples against the existing gold standard IFA to enable a 501(k) submission to FDA.