Real-time PCR Diagnostic Kit for Giardia and Cryptosporidium

描述（由申请人提供）：这项为期两年的提案寻求IDX Labs LLC的小企业支持，继续与弗吉尼亚大学的Houpt博士合作，将贾第虫和隐孢子虫的敏感实时PCR（qPCR）测试带到美国实验室。根据CDC的数据，这些寄生虫每年感染多达200万美国人，我们已经开发了针对这两种感染的强大敏感的qPCR检测方法，并已成功用于孟加拉国和坦桑尼亚的数百名患者。在I期，这些检测试剂盒被转换为隐孢子虫/贾第虫多重检测试剂盒，可检测所有已知的人类亚种，并可在两个主要的qPCR平台（Roche LightCycler和BioRad iCycler）上使用。在第一阶段，我们还确定了最敏感的DNA提取程序中的市售试剂盒，然后通过磁性靶特异性DNA捕获进一步提高PCR检测的灵敏度。该第二阶段申请现在将完成从粪便中提取目标DNA的程序，以获得最高通量，并从我们在坦桑尼亚的研究中心收集验证数据，以准备FDA 501（k）申请，我们预计这将允许强大的市场进入。我们假设，我们的技术将比典型的金标准Merifluor隐孢子虫/贾第虫IFA更敏感，我们将利用第三个决胜PCR检测来解决任何不一致的结果。其次，该测试也将在水样中与EPA的贾第鞭毛虫/隐孢子虫方法#1623进行验证，考虑到市场和对饮用水和娱乐水样中这些原生动物的改进测试的需求。这一应用的创新在于DNA捕获提取技术，这是一个可转换到粪便以外的其他临床标本的平台，以及将抗原检测之外的最先进诊断技术引入资金不足的水传播健康重要性寄生虫感染的概念。 该II期申请将推进我们对贾第虫/隐孢子虫真实的时间PCR试剂盒的开发，该试剂盒将高度灵敏的靶特异性DNA提取与高度优化的多重qPCR测定相结合。在第二阶段，将完成最终的样本到结果试剂盒，并将根据现有的金标准IFA对临床样本进行性能验证，以便向FDA提交501（k）申请。

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.