Development Of Array-based Technologies For Multiplex Detection Of Nucleic Acids And Their Applications In Monitoring Of Genetically Modified Organisms

With the rapid development of genetically modified organisms(GMOs)in the world,more and more genetically modified events have been approved to enter the market,and the composition of the inserted fragments is increasingly complicated and diversified.Meanwhile,illegal circulation of unauthorised GMOs and crops or products mixed with low level of GMOs or ingredients occasionally happen in global market.There is thus an urgent need for highthroughput GMO monitoring methods which are fast,efficient,simple-tooperation and low-cost.However,current GMO detection approaches face several problems and challenges,such as insufficient number of targets that can be detected in a single test,complicated and time-consuming procedures due to separated amplification and detection steps,and lack of portable platforms.Further,the problems that GMO monitoring face are common problems of many other fields in which multiplex detection of nucleic acids are required,such as disease diagnosis,microbial detection,food safety monitoring,and environmental monitoring.To help solve these problems,in this thesis,we apply the strategy of physically isolating individual primers and reactions,and successfully develop a set of multiplex amplification and detection platforms for nucleic acids based on various microarray technologies.We successfully apply these methods in the high-throughput detection of GMOs,and to some extent solve the main problems of the current GMO monitoring field.These platforms also provide new solutions for the whole multiplex nucleic acid detection field.In the first part of this thesis,to increase the target number that can be detected in a single GMO monitoring test,in the first part of this thesis,we take advantage of a highly efficient multiplex chip-PCR strategy that we have previously developed and construct a combined microarray system named MACRO(Multiplex Amplification on a Chip with Readout on an Oligo microarray),which is composed of a PCR chip for the multiplex amplification of 91 target DNA fragments and an oligo microarray for the readout of the amplified DNA fragments.With this system,we show a GMO event coverage of 97% and a limit of detection that is suitable for practical applications.The results of simulated complex samples and blind samples obtained from MACRO are 100% consistent with expectations and the results of independently performed real-time PCRs,respectively.MACRO is the first system that could be applied to effectively monitor the majority of the commercialized GMOs in a single test,and will be of important meaning to the routine monitoring and regulation of GMOs.Then,to reduce the operation steps and detection time of the current methods,in which amplification and detection are separated,in the second part of this thesis,we improve the MACRO platform to simplify the experiment procedure and shorten the reaction time,and develop a one-step platform called FLAC(Fluorescent-Labeled Amplification and analysis on Chip).We employ TaqMan probe technology to fluorescently label the chip PCR products,and develop a simple way to cover the chip after the chip PCR step,so that the fluorescent signals of the chip PCR products can be read directly by a microarray scanner.This platform realizes in situ detection of the chip PCR products,reducing the detection time to less than a half of conventional detection time.This method is also applied to detect multiple GMO targets.We choose several important GMOrelated elements and endogenous reference genes for the test.We successfully detected 19 GMO-related targets in a single rapid test.The preliminary results show that this method has high specificity and sensitivity.In the last part of this thesis,to improve the portability of multiplex detection platforms of nucleic acids,we use the same strategy of physical isolating individual primers and reactions to develop a simple,rapid and low-cost portable platform called CALM(Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids),which combine an integrated capillary array as multiplex micro reactors and visual LAMP(Loopmediated isothermal amplification)technology.In this method,we combine a hydrophobically patterned capillary array and a simple design for sample delivering,enabling simultaneously delivering and separating sample into all of the capillaries very simply.Taking advantage of visual LAMP technology,the test can complete within half an hour to an hour,and the results can be directly observed by the naked eye,without needs for complicated equipments.This platform is easy to operate,with low dependence to equipment and electricity,therefore is expected to be well applied to point of care tests(POCTs)in future.Also,we apply this method to GMO monitoring,in which we successfully detect 8 important GMO-related targets in a single rapid visual test.Monitoring results of simulated samples and real-world samples show high specificity,sensitivity and accuracy.All the results suggest that this platform is very suitable for routine GMO monitoring.In conclusion,in this thesis we take advantage of various forms of microarrays and the strategy of primer and reaction isolation,building up a set of simple and univesal multiplex amplification and detection platforms for nucleic acids.These methods can greatly improve the multiplicity of nucleic acid detection.Moreover,with high specificity and sensitivity,these methods are also simple,flexible and versatile.All these methods are successfully applied to GMO monitoring,which has an urgent need for multiplex nucleic acids detection methods,showing good results and great advantages over conventional methods.Therefore,we strongly believe that these approaches will be widely applied for future routine GMO monitoring.In the mean time,these platforms also provide new solutions for the whole field of multiplex nucleic acid detection.