Development Of Multiplex Nucleic Acids Detection Method Based On Microarray And Its Application In Genetically Organisms Detection

Multiple nucleic acid detection methods have been widely applied in the different fields,such as pathogen detection,gene detection,GMO monitoring,molecular breeding and food-borne and environmental monitoring,because it can detect and analyze multiple targets in a single reaction at the same time.However,the multi-target detection methods face many problems and challenges currently.Take the most widely used nucleic acid amplification technology PCR and LAMP for an example,the multiplicity of amplification targets is limited in a reaction system due to the mutual interference and competition among the primer pairs making the amplification loci unbalanced and the efficiency reduced.What's more,the multiplex nucleic acid detection field also faces some problems,for example,the complicated and time-consuming process due to the separated steps of multiplex amplification and detection,and the lack of portable detection platform.To help solve the above issues,this research develops a multiplex nucleic acid amplification detection platform to solve different problems by the strategy of physical isolation.And take the detection of genetically modified organisms as an example to illustrate,to provide new solutions and ideas for the current multiplex nucleic acid detection fields.First,in order to increase the multiplicity of amplification and integrate the amplification and detection steps,we designed a chip with hydrophilic microwells and hydrophobic channels.Different primer pairs and low melting agarose is pre-fixed in different microwells,making the reaction system is able to solidify after the end of the amplification,and the nucleic acid dye is introducing into channel and then results are visually detected by a home-made,potable UV detector.Comparing with the amplification efficiency of conventional PCR,it shows that our chip platform has the same amplification efficiency.Meanwhile,we detect the different combinations of seven common and important targets of genetically modified organisms and results show that the method has high specificity and flexibility.If our method is further optimized,it can be applied to GMO(Genetically modified organisms)monitoring and other multiplex nucleic acid detection field.Second,in order to develop the portable detection platform ultimately.In this paper,the previous platform,CALM(Capillary Array-based LAMP for Multiple Detection of nucleic acids),developed in our lab is further improved and standardized.Now,the process of fabrication and experiment is easier and robust to achieve.We apply mold to produce PDMS array,and then assemble the capillary array and treat it with hydrophilic or hydrophobicity pattern then anchor the sample-loading adaptor.LAMP reaction system can be loaded in different capillaries simultaneous by just a single pipetting.Due to the constant reaction temperature of visual LAMP technology,the process is easy and less rely on equipment.In this method,8 kinds of transgenic targets are detected quickly and visually without relying on complex professional instruments,and it have high potential to be applied for multiplex nucleic detection in resource-limitation areas and POCT(Point of Care Test).Finally,to make portable multiplex nucleic acid detection platform easier and more stable,we apply the idea of capillary separation and hydrophobicity modification to develop a new capillary array based cassette which have a simple production and sample-loading process.Arrange the capillary in a circle with one end contact the central hole and modify the center hole with hydrophobic.The reaction system can be loaded into different capillaries simultaneously by a single pipetting in the central hole to achieve simultaneous amplification and detection.The preliminary results showed that our method can quickly and visually detect the targets of genetically modified organisms.In summary,to help solve the current problems faced in multiplex nucleic acid detection,in this thesis we develop different methods based on the strategy of physical isolation primers to bypass the fundamental bottleneck problem of low multiplicity in multiplex nucleic acids detection technology.We apply different forms of microarray to develop a set of universal multiplex nucleic acid detection technology,which have high specificity,high flexibility and these methods are also simple,resource-affordable.In these research,GMO monitoring has been successfully applied and showed good results.So We believe that after improve and more research in these methods,they can not only play a practical role in the field of multiplex nucleic acids detection which including GMO monitoring,but also provides some new ideas for the entire fields.