Ultra-sensitive magnetic assays for quantitative detection of nucleic acids witho

描述(申请人提供)：MagneSensors公司的计划旨在开发一种快速、超灵敏的磁性分析方法，在不需要聚合酶链式反应或其他扩增的情况下定量检测核酸。这种新型的三明治杂交检测方法利用了一种精密灵敏的磁性传感器，可以测量杂交到RNA靶标的核酸探针上磁性纳米颗粒标记的磁场。在临床诊断中有许多应用，例如检测引起脓毒症的病原体，但由于污染和其他问题导致的假阳性和假阴性等缺点，扩增的核酸测试一直难以被接受。在直接检测方法上花费了大量的努力，但在临床标本上实现高灵敏度的挑战被证明是令人敬畏的，并限制了应用。磁性检测提供了独特的优势，如高灵敏度、特异性、快速和简单，这是竞争平台很难实现的组合。我们最新一代的台式仪器使用高温超导量子干涉装置(SQUID)，通过小型制冷器进行冷却，能够立即以非常高的灵敏度测量50个分析样品。用户甚至不需要知道冷却，这一直是使用SQUID的一个经常被引用的缺点，并且该仪器非常适合临床诊断。第一阶段的目的是展示磁学检测金黄色葡萄球菌中的核糖体RNA(RRNA)的基准：1)6×104拷贝rRNA(100zmoL)，相当于每毫升样本体积中的H 20 cfu/ml金黄色葡萄球菌，2)金黄色葡萄球菌与表皮葡萄球菌对照的特异性，3)45分钟的检测准备和测量(不包括RNA提取)和4)不分离未结合的磁性纳米颗粒标记。因此，化验方法利用了每种细菌中丰富的rRNA(500-2000个拷贝)。另一个创新方面是使用肽核酸(PNA)探针。这些合成的核酸具有更好的结合、更高的特异性、更高的稳定性、缩短的杂交时间和抵抗酶降解的优点。预计上述目标将在第二阶段得到进一步改进，能够在30至45分钟的总测试时间内检测到单一细菌以及特定的甲氧西林耐药金黄色葡萄球菌(MRSA)。与公共卫生相关：快速、轻松地对核酸进行定量测量而无需放大的能力将打开一系列重要应用的大门。开发一个用于快速、准确和具有成本效益的细菌检测系统的超敏感平台将使传染病的诊断、预防和治疗发生革命性变化。这些感染包括社区和医院获得性感染，如耐药葡萄球菌(MRSA)，这种感染正在上升，并经常导致败血症。

DESCRIPTION (provided by applicant): MagneSensors' program is aimed at developing a rapid, ultra-sensitive magnetic assay to quantitatively detect nucleic acids without the need for PCR or other amplification. The novel sandwich hybridization assay takes advantage of an exquisitely sensitive magnetic sensor that measures the magnetic field from magnetic nanoparticle labels on nucleic acid probes hybridized to RNA targets. There are many applications in clinical diagnostics, such as the detection of pathogens causing sepsis, where amplified nucleic acid tests have struggled to gain acceptance because of drawbacks including false positives and false negatives due to contamination and other issues. Much effort has been expended on direct detection methods but the challenges in achieving high sensitivity on clinical specimens have proven formidable and limited applications. Magnetic detection offers unique advantages such as high sensitivity, specificity, speed, and simplicity, a combination that is very difficult to achieve in competing platforms. Our latest generation bench top instrument uses a high temperature superconducting quantum interference device (SQUID) that is cooled with a small cryorefrigerator and is capable of measuring 50 assay samples in a minute at very high sensitivity. The user needs not even be aware of the cooling, which has been an often-cited drawback for using SQUIDs, and the instrument is well-suited for clinical diagnostics. The Phase I aim is to demonstrate magnetic detection of ribosomal RNA (rRNA) in Staphylococcus aureus with the following benchmarks: 1) 6 x 104 copies rRNA (100 zmol), equivalent to H 20 CFU/ml S. aureus in 1 ml sample volume, 2) specificity of S. aureus vs. S. epidermidis control, 3) 45 minutes assay preparation and measurement (exclusive of RNA extraction) and 4) no separation of unbound magnetic nanoparticle labels. The assays thus take advantage of the abundance of rRNA (500-2,000 copies) in each bacterium. Another innovative aspect is the use of peptide nucleic acid (PNA) probes. These synthetic nucleic acids offer advantages of better binding, increased specificity, higher stability, reduced hybridization time, and resistance to degradation by enzymes. It is expected that the above aims would be much further improved in Phase II, enabling detection down to a single bacterium as well as specific detection of methicillin resistant Staphylococcus aureus (MRSA) in a total assay time of 30-45 minutes. PUBLIC HEALTH RELEVANCE: The ability to quickly and easily perform quantitative measurement of nucleic acids without amplification would open the door to a host of important applications. Development of an ultra-sensitive platform for rapid, accurate, and cost effective bacterial detection systems would revolutionize the diagnosis, prevention, and treatment of infectious diseases. These include community and hospital acquired infections such as staphylococcus resistant to antibiotics (MRSA), which are on the rise and often lead to sepsis.