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

描述（由申请人提供）：MagneSensors的项目旨在开发一种快速，超灵敏的磁分析方法来定量检测核酸，而无需PCR或其他扩增。这种新型的三明治杂交试验利用了一种非常灵敏的磁传感器，可以测量核酸探针上的磁性纳米颗粒标签与RNA靶标杂交的磁场。在临床诊断中有许多应用，例如检测引起败血症的病原体，其中扩增核酸检测由于污染和其他问题导致的假阳性和假阴性等缺点而难以获得接受。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已被证明是艰巨的和有限的应用。磁检测具有独特的优势，如高灵敏度、特异性、速度和简单性，这些优势在竞争平台中很难实现。我们最新一代的台式仪器使用高温超导量子干涉装置（SQUID），用小型冷冻冰箱冷却，能够在一分钟内以非常高的灵敏度测量50个分析样品。用户甚至不需要意识到冷却，这是使用squid的一个经常被引用的缺点，而且该仪器非常适合临床诊断。第一阶段的目标是通过以下基准证明对金黄色葡萄球菌核糖体RNA （rRNA）的磁检测：1)6 × 104拷贝rRNA （100 zmol)，相当于1 ml样品体积中金黄色葡萄球菌的h20 CFU/ml， 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），4）未结合的磁性纳米颗粒标记不分离。因此，这些检测利用了每个细菌中丰富的rRNA（500- 2000个拷贝）。另一个创新方面是肽核酸（PNA）探针的使用。这些合成的核酸具有结合性好、特异性高、稳定性好、杂交时间短、不易被酶降解等优点。预计上述目标将在II期得到进一步改善，能够在30-45分钟的总检测时间内检测到单个细菌以及耐甲氧西林金黄色葡萄球菌（MRSA）的特异性检测。公共卫生相关性：无需扩增而快速简便地进行核酸定量测量的能力将为许多重要应用打开大门。开发一种超灵敏的平台，用于快速、准确和经济有效的细菌检测系统，将彻底改变传染病的诊断、预防和治疗。其中包括社区和医院获得性感染，如耐抗生素葡萄球菌（MRSA），这种感染呈上升趋势，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.