Establishment of a recombinase polymerase amplification combining with lateral flow strip assay for rapid visual detection of Francisella tularensis

ObjectiveTo establish a rapid, sensitive and specific assay for detection of Francisella tularensis by recombinase polymerase amplification and lateral flow (LF-RPA) strip techniques.

MethodsA primer-probe set targeting specific gene tul4 of F. tularensis for LF-RPA assay was designed and the parameters (temperature and time) for the assay were optimized. The sensitivity and specificity of the assay were evaluated.Field feasibility was explored by running the assay with Francisella -spiked blood samples.

ResultsThe optimal temperature and time of the assay were at 40 ℃, and for 20 min. The limit of detection (LOD) of the LF-RPA assay on genomic DNA of F.tularensis was 20 fg/μl, similar to qPCR method targeting the same gene. No cross-reactions with 7 other non-F. tularensis bacteria species was observed,indicating the specificity of the LF-RPA assay. The LOD of the LF-RPA assay for detecting F. tularensis in simulated blood samples was about 460 CFU/ml.

ConclusionThe newly developed assay is a simple, rapid, sensitive and specific method for the detection of F. tularensis with a potentiality of clinical diagnosis of tularemia and detection of Francisella at an outbreak field.