Establishment of an isothermal amplification method for detection of viable Salmonella enterica subsp. enterica Derby

Abstract:

[Background] Salmonella enterica subsp. enterica Derby as a major pathogenic serotype is harmful to the public health.[Objective]To build a rapid and accurate approach for the detection of this serotype.[Methods]An accurate and effective method was built with propidium monoazide (PMA)-recombinase polymerase amplification (RPA) for the detection of active S.Derby.[Results]The assay used RU61_00441 gene as target and designed the primer SD1F/R which correctly identified all the tested strains.The PMA treatment effectively distinguished between viable and dead cells.The limit of detection was calculated to be 761.2 fg/μL for genomic DNA and 45 CFU/mL for bacterial culture.For the detection of the serotype Derby,this assay was not affected by the genomic DNA of background flora (pork,chicken,and beef).Importantly,S.Derby in animal-derived food could be detected at a concentration as low as 3.9 CFU/mL after enrichment for 6 h.[Conclusion]This PMA-RPA method is time-saving and has good sensitivity and specificity,which can provide reference for the future detection of Salmonella.