Developing an Isothermal Solid-Phase Nucleic Acid Amplication and Microarray Plateform for Detecting Respiratory Virus and Enterovirus Infection.

Early diagnosis of infection disease and early identification of specific pathogen play the most important role in the prevention of infectious disease. The respiratory viruses and enteroviruses infection are common in Taiwan. According to the Bureau of National Health Insurance report, the medical expenses for upper respiratory tract infection and enterovirus infection are more than 25.5 billion NTD. The global in vitro diagnostics (IVD) market reach 53 billion USD in 2014. The IVD market in Taiwan is about 3 billion NTD, and the virual detection marker is about 0.9 billion NTD. Biochip is a method developed recently for rapid virus detection. The major drawback of biochip is too expensive. Take the DR. Chip Biotech Inc. for example, they had developed biochips for respiratory and enteroviruses detection, but the market acceptance of those products were not positive because of it high price (1,000〜2,000 NTD/chip). This plan aims to develope a solid phase recombinase polymerase amplification (SP-RPA) platform to simultaneous multiplex isothermal amplify and detect viruses nuclic acid on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens respiratory viruses and enteroviruses using virus RNA. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a 96-wells plastic plate. The SP-RPA could extend the possibility to detect and analyze nucleic acids in a POC testing device. Without the need for the thermocycling process these devices could be simplified and would not require expensive additional equipment and thus reduce the overall diagnostic costs. By targeting multiple genes at once the SP-RPAwould allow to test e.g. for a panel of infectious pathogens associated with a particular clinical manifestation in a single experiment. A successful enzymatic reaction is completed in ＜20 min and results in simultaneous detection of 9 respiratory viruses and 5 enteroviruses. The SP-RPA plateform we developed could be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics.

Project ID:PC10505-0284
External Project ID:MOST105-2622-B182-003-CC2