Digital Nucleic Acid Analysis Based On Loop-mediated Isothermal Amplification

The ability to quantify nucleic acids in complex matrices with high sensitivity and accuracy is central to a wide range of applications from biomedical research to clinical theranostics.Routine nucleic acid amplification techniques such as polymerase chain reaction(PCR)provide high sensitivity for id entification of rare species.However,they suffer from limited specificity in mutation discrimination,compromised precision,high susceptibility of polymerase inhibitors and inability for absolute quantification.A promising alternative is digital nuclei c acid detection(d NAD)such as digital PCR(d PCR)by compartmentalizing single nucleic acid molecules and performing PCR to identify the presence of target molecules for each compartment.d PCR has demonstrated better specificity for rare mutation detectio n,higher precision to enable copy number variation analysis,and capabilities of end-point and absolute measurements.These advantages have catalyzed expanding applications of d PCR to various clinical challenges.Current d PCR technologies have been perfor med using three major designs,reaction chamber arrays with microfluidic control,droplets generated with microfluidic chip,and magnetic beads encapsulated in emulsion.Microfluidic d PCR technologies suffer from drawbacks of relatively high cost,limited scalability,and low throughput.BEAMing provides a more cost-efficient and high-throughput d PCR technology using emulsion encapsulated magnetic beads.Despite the advantages,BEAMing is still subjected to issues such as failure in detecting templates partitioned into droplets without beads,susceptibility to polymerase inhibitors in DNA extracts,and complexity in the workflow and thermocycling amplification.For diagnostic applications in resource limited settings,there remains a persistent need for d NAD technologies with high throughput,technical robustness,simple operation,and low cost.Here we develop a novel d NAD platform,BEAMing LAMP,by combining emulsion microreactors and single-molecule magnetic capture and on-bead loop-mediated isothermal amplification(LAMP).Based on the BEAMing LAMP,a series of application in DNA quantitative analysis have also been developed.The details are as follows:In Chapter 2,a novel d NAD platform,BEAMing LAMP,by combining emulsion microreactors and single-molecule magnetic capture and on-bead loop-mediated isothermal amplification(LAMP)is developed for the first time.In addition,the use of LAMP instead of PCR in emulsion amplification can provide better amplification efficiency and specificity without the n eed for a thermal cycler with an accurate temperature control device.By optimizing and improving each assay condition of this technology,it was successfully applied to the determination of HCV RNA in clinical plasma samples.Not only does the platform pr ovide high contrast,identifiable signals within 1.5 h,it achieves a detection limit of 300 copies/m L and enables absolute quantitation with high accuracy.In chapter 3,taking the mutation of BRAF gene as an example,a novel method of digital nucleic acid mutation analysis was developed based on BEAMing LAMP,which is easy to operate,low cost and amplified at constant temperature.A blocked cleavable LAMP inner primer was designed to increase the time gap between the mutant and wild-type templates at LAMP amplification.This allows for the detection of a small amount of mutant samples in mixed samples,even though the mutant and wild-type templates are present in the sample at a concentration ratio of 1: 10000.Therefore,BEAMing LAMP can provide a power ful tool for the clinical diagnosis of hereditary diseases and a useful platform for nucleic acid mutation detection with high sensitivity and accuracy in research of disease mechanism and personalized early treatment.In Chapter 4,we used Shigella DNA testing as an example to develop a new BEAMing LAMP.A fluorescent Cy5 probe was designed complementary to the inner primer.The Cy5 probe hybridized with the inner primer before the LAMP amplification and was captured by the capture probe s on the magnetic beads after LAMP amplification.Then,the magnetic beads produced strong fluorescence signal s.This design provides a rapid and efficient strategy for the quantitative detection of bacteria.The design of primers is simpler and the need of fluorescent probes is reduced compared to BEAMing LAMP using FIP or LB primers as capture probes.Therefore,the cost of experiment is lower.This platform delivers high-contrast,identifiable,high-fluorescence-positive magnetic beads in 40 min with a limit of detection of 500 copies and absolute quantitation with high accuracy.In Chapter 5,a novel methylation BEAMing LAMP,by combining bisulfate treatment and conversion of the methylation sequence,emulsion microreactors,single-molecule magnetic capture and on-bead loop-mediated isothermal amplification(LAMP)was developed.The methylation BEAMing LAMP can selectively detect a small amount of methylated samples in a 1: 100,000 mixture of methylated and unmethylated sequences within 50 min without being disturbed by a large number of unmethylated sequences.These results indicate that our methylation BEAMing LAMP provides a rapid and efficient method for the detection of methylated nucleic acids and enables an accurate assessment of the methylation level of clinical samples.