Rapid and sensitive detection of the vanA resistance gene from clinical Enterococcus faecium and Enterococcus faecalis isolates by loop-mediated isothermal amplification.

ABSTRACT

OBJECTIVES: Vancomycin resistance in Enterococcus spp., mediated mainly by the vanA resistance gene, has become a major health concern as it has spread worldwide. Therefore, a rapid method is urgently required to detect the vanA gene for timely and appropriate antimicrobial control of resistant Enterococcus infections. METHODS: The loop-mediated isothermal amplification (LAMP) assay was optimised for vanA detection in Enterococcus spp. isolates. RESULTS: The LAMP primer set designed in this study could reliably recognise seven distinct regions of the vanA gene and amplify the gene within 25min at an isothermal temperature of 65°C with high specificity. The sensitivity of the optimised assay was high, with a detection limit for vanA as low as 100pg/µL, which is 100-fold more sensitive than the PCR assay. A special advantage of this optimised LAMP method is that the vanA gene could be detected directly from clinical specimens. CONCLUSION: This optimised LAMP assay has great application potential for efficient detection of vanA in clinical diagnosis and epidemiological studies.