Rapid, on-chip, multiplexed detection of sepsis-causing organisms from blood samples

这项建议将Biogene在快速聚合酶链式反应开发、仪器设计与制造和软件开发方面的专业知识与赫尔大学在微流控芯片设计和优化以及具有真实世界接口的设备制造方面的成熟记录结合在一起，例如犯罪现场或护理地点。该小组将共同建立一个快速、便携、高精度的系统，能够从血液样本中提取病原体DNA，以确定具有相关亚型特异性的多种细菌物种的存在，例如葡萄球菌的凝固酶状态。所有用于DNA提取、PCR扩增和分离的试剂都将预装在设备中。该仪器的操作和稳定性基于赫尔团队发表的工作。Biogene拥有一套经过验证的光学系统，能够分离10多种染料，当这项技术与使用赫尔公司的微流控设备对聚合酶链式反应产物进行电泳性分离的能力相结合时，新仪器将能够区分数千个不同的PCR片段。该检测系统基于由大量差异标记产物产生的荧光信号的光学去卷积。这种方法能够检测到比目前常规测序仪所能检测到的更多的目标。在这项概念验证研究中，我们打算表征至少10个特定因素，即8个生物体和2个物种特定因素；然而，预计将实现相当大的多重水平。血液被广泛认为是病原体核酸扩增的“困难介质”，这既是因为存在广泛的聚合酶链式反应抑制剂，也是因为存在大量过量的人类基因组DNA。血液样本将首先经过处理，以溶解红细胞并促进细菌细胞的收集，首先通过过滤，然后回收浓缩的细菌细胞，以便为DNA分析提供起始材料。其目的是制造一种适合处理微升容量的平台，因为这将使通过毛细管采集血液的新生儿和儿童能够检测到败血症。使用少量血液的另一个好处是降低了整个单位的复杂性和成本。聚合酶链式反应系统的重点将是最大限度地提高灵敏度和速度。在进行实时聚合酶链式反应格式测试和多重混合测试之前，将在大量的计算机模拟之后确定每个目标的最佳聚合酶链式反应引物对。2010年，将从赫尔和东约克郡患者的败血症致病微生物名单中挑选出最常见的微生物。为交付该系统将解决的其他关键因素是：有效的样本界面和DNA提取的自动化；将超快速聚合酶链式反应装置和光学系统整合到微流体平台上；以及定制的控制软件，包括数据存储和用户友好的操作员显示器。系统的每个部分将在分析本身的设计以及使用添加了目标基因组等价物的捐献者的血液样本构建概念验证单元的过程中进行广泛测试，以测试功能。对全功能设备的测试将在以下患者的血液中完成：i)新出现的具有典型败血症症状的急诊急诊室(n=50)或ii)重症监护病房(n=15)。为临床评估而获得的病理报告将给出一个比较的“金标准”。赫尔和东约克郡NHS信托基金的所有顾问W·汤恩德博士(急诊医学)、S·贝内特博士(心胸麻醉和重症监护)和R·梅格博士(微生物学家)将担任临床顾问团队。适当的认证，例如CE标志和IVD认证(98/79/EC)，将在临床试验完成后申请。

This proposal brings together BioGene's expertise in development of rapid PCR, instrument design & manufacture andsoftware development, with the University of Hull's proven track-record in microfluidic chip design & optimisation, andfabrication of devices with real world interfaces, e.g. scene of crime or point of care. Together the group will build a rapidand portable, highly-accurate, system capable of extracting pathogen DNA from a blood sample, to determine the presenceof multiple bacterial species with relevant sub-type specificity, e.g. coagulase status for Staphylococci. All reagents for DNAextraction, PCR amplification and separation will be preloaded in the device. The operation and stability of this instrumentare based on published work from the Hull team.BioGene have a proven optical system capable of separating more than 10 dyes, and when this technology is coupled withthe ability to electrophoretically separate PCR products down to 2 base pair resolution, using Hull's microfluidic device, thenew instrument will be able to discriminate 1000s of distinct PCR fragments. The detection system is based on opticaldeconvolution of the fluorescence signal generated by the multitude of differentially-labelled products. This method enablesthe detection of many more targets than is currently possible with conventional sequencers. In this proof of concept studywe intend to characterise at least 10 specific factors, i.e. 8 organisms and two species-specific factors; it is anticipatedhowever that a considerably greater level of multiplexing will be achieved.Blood is widely recognised as a "difficult medium" for pathogen nucleic acid amplification, both due to the presence of awide range of PCR inhibitors and the vast excess of human genomic DNA present. The blood sample will first be treated tolyse red blood cells and facilitate collection of bacterial cells, initially by filtration, followed by recovery of the concentratedcells to provide the starting material for the DNA analysis. The aim is to produce a platform suitable for handling microlitrevolumes as this would allow detection of sepsis in neonates and children where blood collection is via capillary tubes. Anadditional benefit of using smaller volumes of blood is a reduction in complexity and cost of the overall unit.The focus of the PCR system will be to maximise sensitivity and speed. Optimal PCR primer pairs for each target will beidentified after extensive in silico modeling before being tested in a real-time PCR format and for compatibility in a multiplexmix. A selection of the most common organisms will be chosen from a list of sepsis-cuasing organisms in Hull & EastYorkshire patients during 2010. Other key factors that will be addressed to deliver the system are: an effective sampleinterface and automation of DNA extraction; incorporation of the ultra-rapid PCR unit and optical system onto a microfluidicplatform; and bespoke control software that includes data storage and user-friendly operator display.Each part of the system will be tested extensively during design of the assays themselves as well as construction of theproof of concept unit using blood samples from donors spiked with genome equivalents of the targets in order to testfunctionality. Testing of the fully-functional device will be completed on blood drawn from patients: i) newly-presenting atAccident & Emergency with classic symptoms of sepsis (n=50) or ii) on the Intensive Care Unit (n=15). The pathologyreports, obtained for clinical assessment, will give a "gold-standard" for comparison. Dr W Townend (Emergency Medicine),Dr S Bennett (Cardiothoracic Anaesthesia and Intensive Care) and Dr R Meigh (Microbiologist), all Consultants at Hull &East Yorkshire NHS Trust, will act as a clinical advisory team. Appropriate certification, eg. CE marking and IVDcertification (98/79/EC), will be applied for once the clinical trials have been completed.