Rapid isothermal detection assay: a probe amplification method for the detection of nucleic acids

Simple, accurate, and stable diagnostic tests are essential to control viral infectious diseases such as avian influenza virus. The current technologies are often inaccessible to people who need them, mainly because of the specialized equipment and the need for highly trained technologists. Here, we describe a rapid isothermal nucleic acid detection assay (RIDA) that can be used to detect both DNA and RNA targets. Using chemically modified probes, we designed a lateral-flow (LF) immunoassay that can be used in combination with RIDA for equipment-free nucleic acid target detection. RIDA is a "probe amplification" assay that uses the single-strand nicking activity of restriction nicking endonucleases to repeatedly cleave synthetic probes hybridizing to the same target sequences. In the RIDA-LF combined assay, chemically labeled probes are covalently conjugated to magnetic microbeads, which is propitious to separate cleaved probes from the reaction solution. The cleaved probes in the solution are then detected with an LF immunoassay. The real-time assay shows that RIDA is able to specifically detect target sequences in 5 to 15 min. The RIDA-LF combined assay can specifically detect nucleic acid targets without sophisticated equipment. In this report, our data suggest that RIDA is a flexible simple assay that could be applied for point-of-care detection. The modified-RIDA described in this report further extends the application of this technology.