Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-mediated isothermal amplification (LAMP) for a rapid detection of the presence of vanA gene, a critical component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62°C and with 2mM MgSO4. The detection limit of the DNA template was 80pg and LAMP amplicons were detected within 40min; thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxy naphthol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection.
Bibliographical noteFunding Information:
This work was also supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs , HI12C1251-00013 . We thank So-ri Jong and In Hae Kim for their excellent technical assistance. We are also thankful to Dr. Yonggen Cho (Yonsei University, South Korea) for helping us design LAMP primers.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Microbiology (medical)