Analysis and detection of RNA viruses require rapid, robust, and affordable amplification of long viral RNA (vRNA) strands. Under optimized conditions, in vitro transcription (IVT) meets these criteria; phage polymerases can repeatedly create RNA from complementary DNA templates. Because the IVT is a relatively simple reaction that is conducted at constant temperature, it offers a distinct advantage over other amplification methods such as polymerase chain reaction (PCR). In this paper, we conducted IVT reactions using both immobilized and free DNA templates to show that robust amplification of long strands of influenza A RNA from bead immobilized templates is both possible and advantageous, given proper protocol and reaction conditions. By immobilizing the DNA template on beads, one can better control solution conditions by replenishing the reaction solution while recycling the DNA template. The reaction was then transferred to a microreactor, where solution conditions were carefully controlled by flow rate through a packed bead bed. This method creates a simplified and efficient amplification method for long RNA strands with potential for automation, which can ultimately be transferred to a field deployable device.

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