Establishment and Evaluation of a Real-time Recombinase-aid Amplification Assay for Rapid Detection of Tetracycline Resistance Gene tet(A)

Antibiotic resistance genes (ARGs), as a new type of environmental pollutant, pose a serious threat to ecology and human health, and have attracted the attention of many disciplines. To achieve rapid and accurate detection of ARGs, and improve the ability of environmental monitoring and treatment, this study established a rapid detecting method of tetracycline resistance gene tet(A) based on recombinase-aided amplification (RAA) technology. The detecting method takes the conserved region of tet(A) gene sequence as the target sequence, designs its RAA primers, screens and optimizes RAA primer probes, and establishes a rapid detecting method for a fluorescencing tet(A)-RAA. Results show that the tet(A)-RAA method can specifically realize effective amplification of the tet(A) gene fragment within 30 minutes under the constant temperature of 39 ℃, and has no cross-reaction with non-resistant bacteria. 18 environmental samples were detected by this method, and the positive rate was 100%, which was consistent with the detection result of qPCR method. The tetracycline resistance gene tet(A) detection method established in this study has high sensitivity, good specificity, short reaction time and simple operation, and can be used for the rapid detection of the resistance gene tet(A). It also provides a reference for the development of other rapid detection methods for antibiotic resistance genes.