Rapid miRNA detection with one-pot isothermal rolling circle amplification

This study is led by Professor Jue Ruan (Chinese Academy of Agricultural Sciences, Shenzhen, China). The authors developed a one-pot isothermal detection method based on rolling circle amplification (RCA) for rapid and specific detection of miRNAs and short RNA fragments. This method integrates target identification, linear rolling circle amplification (linear RCA), primer generation in one tube, and the detection time could be shortened within 1 h. Moreover, ROA is highly sensitive, achieving a limit of detection (LOD) of 6pM. The results are easily read visually and verified to be as accurate as RT-PCR.

The principle of ROA is as follows. First, a circular probe for the miRNAs is designed to be tested. During detection, the circular probe binds specifically to the target miRNAs at 57°C-60°C. Generally, rolling circle amplification is performed under the action of Bst polymerase. Here we also artificially made a U-nick reactions by adding a certain proportion of dUTP and a restriction endonuclease that recognizes U. In this case, the newly generated DNA single strand in the reaction system will be interrupted by UDG, and continues to trigger new rolling circle amplification as a new primer, which will promote the assays to achieve exponential amplification in a short time. At last, we use SYBR Gold as an indicator by rapidly detecting the fluorescent signals. ROA can amplify templates from picogram (pg) level to microgram (μg) level within 1 hour. ROA showed excellent adaptability when testing miRNAs of different lengths from 18 to 24 nt. In the specificity test, ROA is found to be sensitive to single nucleotide mismatches, especially at the 3' end. Compared with other UDG-aided RCA, ROA has better specificity.

We further explore the detection ability of ROA for RNA viruses-;-;MS2 phage. It is found that ROA can effectively detect the target RNA of MS2 phage even without removing the host. It demonstrate the clear potential of this ROA assay for the convenient and fast detection of viruses.