Colorimetric monitoring of rolling circle amplification for detection of H5N1 influenza virus using metal indicator

• A procedure for colorimetric monitoring of HRCA via HNB as metal indicator was established.
• The system can be used either as a quantitative biosensor or a qualitative system using naked eye.
• HRCA reaction based on PLP and Bst DNA polymerase led to signal amplification (DL: 28 fM).
• Replacement of the heat shock with pH shock inhibits the possible DNA breaks.

A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg2+ in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28  fM.