A Simple and Rapid Bst Polymerase-Catalyzed Isothermal Exponential Amplification Reaction for Multiplex Detection of MicroRNAs

Bst polymerase is a large fragment of DNA polymerase I with strong strand displacement activity and broad substrate adaptability. It is one of the most commonly used enzymes in developing isothermal DNA amplification reactions for trace nucleic acid detection. Although many of these methods are ultrasensitive and specific, they often suffer from complex primer design, multienzyme dependency, and complicated amplification systems relating to unexpected side reactions and false reported signals. In this work, we designed a simple, rapid, and accurate Bst polymerase-catalyzed isothermal exponential amplification reaction (named BIEAR) for multiplex detection of microRNAs and tried it in diabetic retinopathy (DR) screening and progression risk assessment. The BIEAR system relies solely on one DNA hairpin triggering probe, one double-stranded DNA signal probe, and one type of Bst polymerase to drive bidirectional strand extension and displacement reaction cycles, which produce exponentially amplified fluorescence signals quantitatively dependent on the target microRNAs. The system has achieved pM-graded detection limits in individual and multiplex detection of miR-21 and miR-93, and the reaction time for detecting miR-93 is only 10 min. It exhibited high accuracy with 99.20-105.6% recovery rates in measuring miR-21 in human plasma samples. We have also applied BIEAR to detect concentrations of blood circulating miR-21 and miR-93 and combined them as an indicator for screening DR, achieving a diagnostic sensitivity of 93.80%, a specificity of 84.80%, and an area under the curve of 0.952. This novel method provides a simple, convenient, and accurate approach for potential molecular diagnosis such as early screening of DR.