Development of a real-time multiple cross displacement amplification assay for rapid and highly sensitive detection...

The aim of this study was to develop a method based on multiple cross displacement amplification (MCDA) and real-time fluorescence technique for rapid, highly sensitive and specific detection of Streptococcus pyogenes (Group A Streptococcus, GAS). A set of 10 primers targeting the speB gene of GAS was designed for the MCDA reaction. According to the principle of real-time MCDA detection, the core primer was further modified with a restriction endonuclease recognition sequence, a fluorophore, and a quencher. The optimal reaction temperature for the assay was determined based on the performance of the MCDA amplification products. The detection limit was evaluated using tenfold serial dilutions of GAS genomic DNA templates. To assess specificity, the assay was tested using genomic DNA from 3 GAS strains and 29 non-GAS strains. To evaluate clinical application of the GAS real-time MCDA assay, 56 clinical samples were analyzed and compared with the lateral flow biosensors (LFB) method and PCR. The GAS real-time MCDA method was performed using a fluorescence instrument at 63℃ for 40 min. The method demonstrated high sensitivity, with a detection limit of 50 fg, and showed no cross-reactivity with other pathogens. The GAS real-time MCDA assay described here offers a new and valuable diagnostic tool for the reliable and rapid detection of GAS.