| Viruses are the most abundant, rapidly evolving and diverse biological entities, and are responsible for numerous infectious disease. Recent examples of viral epidemics range from the common cold to SARS, as well as recent Ebola outbreaks in West Africa. Epidemics of virus diseases continue to pose a threat to public health despite the advances of medicine. This is mainly because RNA viruses are the most rapidly evolving organisms on earth, which enables them to escape immune systems, resist treatments and switch between hosts. At the same time the custom virus detection metholds are usually unable to meet the high throughput requirments due to the low detection efficiency; therefore, it presents the urgency and importance of studying the fast virus precise quantitative and rapid detection methold. Droplet microfluidics technology has become the hot spot of microfluidic research in recent years for the high-throughput, low cost and easy fabricate. It has unique advantages in terms of precise quantitative and rapid detection of biological particles. Based on this, we developed a high-throughput drop-based microfluidic system, in order to accurate detect and quantify viruses and other biological samples will dramatically accelerate viral study, thereby enabling quicker therapeutic decisions as well as decreasing the development time for vaccines and antiviral drugs.Firstly, in perspective of micro and nanofluid flow, the basic theory of two-phase flow in microchannel and the droplet formation, fusion and sorting mechanism in the perspective of micro and nano-scale is analyzed. A new two-phase flow dynamics model to analysis of the electric field and flow field, two-phase flow interface is established. The droplets microfluidic and dielectrophoresis technology has been combined to achieve a high throughput biological particle manipulation and accurate sorting. The development of microfluidic technology and the advantages of using microfluidic platform for biochemical analyzes is reviewd, including compare the advantages based on single-phase flow microfluidic platform and droplet-based microfluidic platform. We analyzed the droplet microfluidics and the significance of the development in high-throughput, high accuracy of the biological sample sampling platform establish theoretical principle for the future bioapplication.In order to fast and precisely quantify the target rare mutant RNA molecules from a pool of millions of other RNA templates. Based on drop-based microfluidic system, using 30 nm murine noroviruses(MNV) as a model system, a digital one-step RT-PCR in picoliter drop-based microfluidic system is first present to compartmentalize, amplify, count and isolate single target RNA viruses, which allows us to precisely measure the concentration of viruses at a frequency of 1-2 k Hz per second, and acquire their genomic information one by one. A new microfluidic chip is fabricated that contains a flow-focusing drop maker with a cross section of 25um^2 to produce monodisperse aqueous drops with only 12.5 p L volume. To determine the concentration of viruses by analyzing the number of true negatives or dark drops(N-)and true positives or bright drops(N+)shown in the detection program, using Poisson statistics. Overall, these data show that d RT-PCR offers a very low absolute detection limit for individual RNA molecules.Additionally, based on the quantification of the rare RNA molecules, the viral infectivity measurement is a core procedure to block the life process of virus, and a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidic system with a picoinject step has been developed. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification that fluoresces in the presence of replicated target viruses, and quantified using a custom-built high-throughput drop reader to determine the number of viral infections. Using murine noroviruses(MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. The drop-based assay can be modified for other viral species based upon two critical properties of the virus: the burst size, Bs, and the ratio of viral genomes to PFU, Rg. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests.Finally, recombinant mutations are very infrequent, they have the potential to generate dangerous, virulent strains. A DBM technique is developed that addresses the limitations of current assays for characterizing and quantifying rare genomes: single templates are isolated, individually amplified in drops, and then placed in microwells for processing. By detecting, sorting, and consensus sequencing the amplicons, we unambiguously determined both recombination frequency and loci. The new DBM technique id demostrated by accurate characterization of recombinant progeny from two RNA murine norovirus(MNV) strains, MNV-1 and WU20, by co-infecting a murine RAW 264.7 macrophage culture. This platform decreases the artifacts produced during RT, PCR amplification, and sequencing to an arbitrarily low value. The new approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification. |
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