Development of a polymer-based point-of-care diagnostic in primary care for targeted detection of bacterial DNA in patient samples

我们的目标是满足对直接位于诊所的护理点诊断的未满足需求，该诊断可以在两小时内识别样本中的细菌并产生颜色变化，为临床医生提供细菌感染正在进行的信息，并迅速适当地进行治疗。该诊断装置将由预分散在PCR主混合液中的DNA响应性胶体颗粒（如共聚物胶乳）组成，该PCR主混合液含有DNA聚合所需的所有试剂。为此，临床医生将添加从患者收集的样本，例如血液或尿液。如果样品中存在细菌DNA，则其将通过PCR扩增以形成DNA聚合物。产生的DNA将与响应颗粒相互作用并诱导沉降，产生颜色变化。已经合成了响应于细菌DNA聚合物的颗粒，所述细菌DNA聚合物使用rRNA合酶的通用引物扩增。这个博士项目将专注于优化PCR反应，以确定特定的细菌病原体，如H。pylori、S. aureus，C.空肠，并在装置中测试它们，以分别识别例如胃溃疡、败血症和食物中毒的病原体。然后，该项目将继续确定抗生素耐药性基因，以PCR为目标，以开发传感器，不仅可以指示样本中是否存在细菌，还可以指示哪些抗生素对特定感染无效。这将通过选择识别例如RND外排泵的引物来完成，RND外排泵赋予对一系列常见抗生素的抗性。通过以这种方式开发该设备，我们设想了一种可以识别样品中的高细菌负荷、样品中最常见的病原体以及细菌携带的任何抗生素抗性基因的管阵列

We aim to meet an unmet need for a point of care diagnostic situated directly in the clinic that can identify bacteria in a sample within two hours and produce a colour change, providing information to the clinician that a bacterial infection is ongoing and treat rapidly and appropriately. This diagnostic device will consist of DNA-responsive colloidal particles (such as copolymer latexes) pre-dispersed in a PCR master mix solution containing all of the reagents necessary for DNA polymerisation. To this the clinician will add a specimen collected from a patient such as blood or urine. If bacterial DNA is present in the sample it will be amplified by PCR to form DNA polymers. The resultant DNA will interact with the responsive particles and induce sedimentation, producing a colour change. Particles that respond to bacterial DNA polymers, amplified using universal primers to rRNA synthase, have been synthesised. This PhD project will focus on optimising the PCR reaction to identify specific bacterial pathogens such as H. pylori, S. aureus, C. jejuni and test them in the device to identify causative agents of, for example, stomach ulcers, sepsis, and food poisoning respectively. The project will then move on to identifying antibiotic resistance genes to target with PCR in order to develop the sensor to give indications of not just whether bacteria are in the sample, but what antibiotics would be ineffective for the specific infection. This would be done by choosing primers that recognise, for example, RND efflux pumps that confer resistance to a range of common antibiotics. By developing the device in this way, we envisage an array of tubes that can identify high bacterial load in a sample, the most common pathogen(s) in the sample and any antibiotic resistance genes carried by the bacteria