Development and application of loop-mediated isothermal amplification assays on rapid detection of various types of staphylococci strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various Staphylococcus strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets: 16S rRNA, femA and mecA. Forty-one reference strains, including various species of gram-negative and -positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65°C for 45min, with detection limits at 100fgDNA/tube and 10CFU/reaction for 16S rRNA, 100fgDNA/tube and 10CFU/reaction for femA, 1pgDNA/tube and 100CFU/reaction for mecA, respectively. Application of LAMP assays was performed on 118 various types of Staphylococcus isolates, the detection rate of LAMP assays for the 16S rRNA, femA and mecA was 100% (118/118), 98.5% (64/65) and 94.3% (66/70), and the negative predictive value (NPV) was 100%, 98.1% and 92.3% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various Staphylococcus strains, and undoubtedly, the rapidness, technical simplicity, and cost-effectiveness of LAMP assays will demonstrate broad application for bacteriological detection of food-borne methicillin-resistant Staphylococcus (MRS) isolates. ► We established 3 LAMP detection methods for mecA, femA and 16S rRNA of Staphylococcus. ► The sensitivities of the LAMP assays were 10–1000 times higher than PCR. ► The LAMP assays were applied to 118 various Staphylococcus strains. ► The detection rates of 3 LAMP assays were 100%, 98.5% and 94.3%.