Establishment and Application of Fluorescent Recombinase-aided Amplification Method for Pantoea stewartii subsp. stewarii Detection

Objective The study was conducted to develop a rapid, sensitive and specific assay for detection of Pantoea stewartii subsp. stewarii based on fluorescent recombinase-aided amplification(RAA).

Method The pst-SglmS sequence of P. stewartii subsp. stewarii was used as target sequence, and the specific primers and probes were designed and synthesized to establish a fluorescent RAA assay for rapid detection of P. stewartii subsp. stewarii. The sensitivity and specificity of the fluorescent method was detected. Simulated samples and actual samples were used to evaluate the reliability of fluorescent RAA, with the SN/T 1375-2004 as standard for verification.

Result A fluorescent RAA for detection of P. stewartii subsp. stewarii was established, which could detect P. stewartii subsp. stewarii specifically at 39℃ within 20 min. The sensitivity of the fluorescent RAA was 280 fg/μL. With the established method, 6 simulated samples were positive and 24 real samples were negative with the same results as SN/T 1375-2004.

Conclusion The fluorescent RAA method for detection of P. stewartii subsp. stewarii is rapid, sensitive and specific, which is proved to be a great choice for site monitoring of P. stewartii subsp. stewarii.