Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

Abstract

We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 10 ⁴ or 2.3 × 10 ⁶ CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 10 ⁶ CFU, but PCR was negative at the level of 2.3 × 10 ⁷ CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 10 ³ or 2.3 × 10 ⁶ CFU, whereas 2.3 × 10 ⁵ or 2.3 × 10 ⁷ CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.