Rapid, Near Patient Nucleic Acid Testing for Congenital Cytomegalovirus (CMV) Infection

摘要 先天性巨细胞病毒(CMV)感染的快速近患者核酸检测 PA-14-088(直达第二阶段) 在美国，普遍的新生儿听力筛查是强制性的，并在产后进行 医院环境。目前基于生理学的听力测试无法发现迟发性婴儿听力损失。 由先天性巨细胞病毒(CMV)感染引起。抗病毒治疗可改善巨细胞病毒相关性 如果婴儿在出生后很快就开始听力损失；因此，一种补充筛查方法 建议识别先天性巨细胞病毒感染的新生儿，以便及早发现和 干预和预防巨细胞病毒相关的听力损失和发育迟缓。一项核酸测试已经完成 描述了使用比以下方法更敏感的唾液样本来筛查新生儿先天性CMV感染 新生儿筛查实验室传统上使用的干血斑点。尽管存在这样一种 测试，公共新生儿筛查计划没有必要的基础设施来测试新生儿唾液 样本。Baebies的使命是通过为新生儿筛查带来新的测试来拯救新生儿的生命。至 解决了实施CMV筛查的当前挑战，Baebies开发并成功测试了 基于实验室数字微流控平台的巨细胞病毒核酸检测。绕过这些限制 并将这一检测带到医院和分娩中心，我们将添加 为我们的近距离患者检测仪器提供功能，从而能够通过聚合酶链式反应进行单一样品分子检测。 我们之前设计了一种盒上聚合酶链式反应方法，通过充分结合样品制备和检测来检测CMV 使用数字微流控技术在同一盒上进行聚合酶链式反应检测巨细胞病毒。有了这种化验，我们 在50微升中仅能检测到5份CMV拷贝。我们的系统面临着200个未识别和 存档的唾液样本；将这些样本和结果与标准的CMV聚合酶链式反应检测结果进行比较。 我们成功地检测出15/17份CMV阳性样本。我们提高了化验的灵敏度，并再次 对我们的系统提出了挑战，从宾夕法尼亚大学出生的新生儿身上收集了130份唾液样本 阿拉巴马州伯明翰(UAB)；所有样本都被正确鉴定。 在这个直接到第二阶段的项目中，我们将通过开发新技术来扩展我们现有的工作 包括盒式加热器和干试剂，使我们的发现者平台能够进行CMV 在医院或诊所使用一次性墨盒和唾液样本进行检测。这个 FINDER仪器正处于单样本生化新生儿筛查的最后开发阶段。 一旦技术开发出来，我们将提高DNA提取效率，进一步缩短见效时间， 并对化验结果进行分析验证。然后，我们将执行一项小型方法比较研究，使用 在两个地点(UAB和Duke)各收集250份回顾性和前瞻性的新生儿唾液样本 大学)。一种近患者新生儿生化和分子联合检测筛查平台 有能力为新生儿筛查引入新的有针对性的通用单一样本测试。

ABSTRACT Rapid, Near Patient Nucleic Acid Testing for Congenital Cytomegalovirus (CMV) Infection PA-14-088 (Direct to Phase II) Universal newborn hearing screening is mandated in the United States and is performed in the postpartum hospital setting. Current physiology-based hearing tests are unable to detect late-onset infantile hearing loss caused by congenital cytomegalovirus (CMV) infection. Antiviral treatment can ameliorate CMV-associated infant hearing loss if initiated quickly after birth; a complementary screening approach has therefore been suggested to identify newborns with congenital CMV infection in order to facilitate early detection and intervention and prevent CMV-related hearing loss and developmental delays. A nucleic acid test has been described to screen newborns for congenital CMV infection using saliva specimens that is more sensitive than the dried blood spots traditionally used by the newborn screening laboratories. Despite the existence of such a test, public newborn screening programs do not have the infrastructure necessary to test newborn saliva samples. Baebies' mission is to save the lives of newborns by bringing new tests to newborn screening. To address the current challenges to implementing CMV screening, Baebies developed and successfully tested a nucleic acid assay for CMV on a laboratory-based digital microfluidic platform. To bypass the limitations inherent to newborn screening laboratories and bring this assay to hospitals and birthing centers, we will add functionality to our near patient testing instrument to enable single sample molecular testing via PCR. We previously designed an on-cartridge PCR assay to test for CMV by fully integrating sample preparation and PCR on the same cartridge for the detection of CMV using digital microfluidic technology. With this assay, we are able to detect as few as 5 copies of CMV in 50 µL. Our system was challenged with 200 de-identified and archived saliva samples; these samples and the results were compared to the standard PCR assay for CMV. We successfully detected 15/17 positive CMV samples. We improved the sensitivity of the assay and again challenged our system with 130 prospectively collected saliva samples from newborns born at the University of Alabama at Birmingham (UAB); all the samples were identified correctly. In this Direct to Phase II project, we will expand upon our existing work by developing new technology including on-cartridge heaters and dried reagents that would enable our FINDER platform to perform CMV testing in a hospital or clinic setting using single use cartridges and readily obtainable saliva samples. The FINDER instrument is in the final stages of development for single sample biochemical newborn screening. Once the technology is developed, we will improve DNA extraction efficiency, further reduce time to results, and perform an analytical validation of the assay. We will then perform a small method comparison study using 250 retrospective and prospectively collected neonatal saliva samples at each of two sites (UAB and Duke University). A near patient newborn screening platform for combined biochemical and molecular testing has the ability to introduce new targeted and universal single sample tests for newborn screening.