Magnetic Nanoparticles Based Trace Nucleic Acid Detection Method

Nucleic acid detection is of great significance for early diagnosis,treatment and prognosis of diseases.However,most of the current mainstream molecular detection technologies require special sample storage and transportation conditions,expensive instruments and professional technicians,and can only be used in relatively developed cities,central hospitals and medical institutions.It is hard to do nucleic acid detection in lower level medical institutions,especially in the vast rural areas and developing countries.In addition,with the development of nanotechnology,magnetic nanoparticles are widely used in the field of molecular diagnosis due to their magnetic conductivity energy.In the presence of an external magnetic field,magnetic nanoparticles can be used to separate,purify and detect nucleic acid molecules from samples.The application of magnetic nanoparticles has obvious advantages,such as no need for centrifugation,easy to realize automation and high throughput detection.Furthermore,visual detection of nucleic acid can be realized by applying the irreversible magnetic nanoparticle mediated aggregation of magnetic nanoparticles.Aiming at some technical bottlenecks existing in current nucleic acid detection technologies and applying some excellent characteristics of functional magnetic nanoparticles,the main contents of this research include the following aspects:First of all,RNA-based nucleic acid detection normally will incur huge costs in sample storage and transportation due to the influence of RNA's easy degradation characteristics.Although the dry blood spot method can solve the problem of sample preservation,the current extraction method is time-consuming,laborious and difficult to apply to automation.In this project,we propose a method to extract RNA from dry blood spot based on magnetic nanoparticles.The method drips collected blood onto a specific paper sheet,and then puts the collected blood and desiccant together into a sealed bag for storage.After blood components are adsorbed on paper sheets and dried,RNA is extracted by magnetic nanoparticles,then NGS library for immune-repertoire constructed and sequencing are carried out.Through the optimization of extraction conditions,it was found that the best effect was obtained by using oligod T magnetic beads to extract RNA from dry blood spot.The method reduces the dependence on centrifuge and other equipment in the extraction process,which can realize automatic extraction and detection of nucleic acid in the cassette.Secondly,aiming at the problem that nucleic acid detection has strict requirements for equipment and site,we propose a solution of nucleic acid visual detection based on magnetic nanoparticles mediated aggregation and hyper-branched rolling circle amplification.The solution uses irreversible aggregation of magnetic nanoparticles with long amplification products to under magnetic field.The method simplifies the complex temperature control system required for nucleic acid amplification and the gel imaging system required for nucleic acid detection,enables the equipment to be miniaturized,the detection to be automated,and the result is visible to naked eyes.