A Programmable Nucleic Acid Scissor Fluorescence Biosensor Based on the Bst-Ni Endonuclease for Detection of the Influenza a (H1n1) Virus

Abstract

Timely and accurate diagnosis of influenza virus is essential to prevent the spread of disease and to select an appropriate treatment strategy. Herein we report the development of a novel fluorescence biosensor for detection of the H1N1 virus based on the Bst-NI endonuclease, dually-blocked RNA scissor (S) strands, and FAM-ssDNA-Q reporter (R) strands. The S strand contains a short (5 nt) sequence for Bst-NI recognition and sequences on both sides of the cutting site, which are closed by two locking strands. The R strand is complementary to the intermediate sequence of the S strand, including the Bst-NI cutting site domain and partial adjacent sequences. Only the presence of two specific RNA fragments of the target influenza virus can fully de-block the S strands, which then hybridize with the R strands, followed by cleavage via Bst-NI-catalyzed hydrolysis, thereby generating the fluorescence signal. The biosensor was sensitive to the H1N1 virus at 500 fM and was highly specific to the target sequence. The proposed biosensor provides a convenient method for reliable detection of the H1N1 virus.

Keywords: H1N1, Bst-NI endonuclease, Primer exchange reaction, Fluorescent biosensor