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. 2023 Nov 6;23(1):329.
doi: 10.1186/s12866-023-02944-4.

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye

Guiling Chen #  1 , Lulin Chen  2 , Sisi Lin  2 , Congzhu Yang  1 , Huanlin Liang  1 , Kuang Huang  2 , Zhusheng Guo  3 , Fei Lv  4
Affiliations

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye

Guiling Chen et al. BMC Microbiol. .

Abstract

The emergence of tigecycline-resistant tet(X2/X3/X4/X5) genes poses a new threat to the efficacy of anti-infective therapy and the safety of our food and environment. To control the transfer of such genes, a sensitive and rapid molecular method is warranted to detect tet(X2/X3/X4/X5) genes in clinical isolates. Herein, we established a loop-mediated isothermal amplification (LAMP) assay to rapidly detect tet(X2/X3/X4/X5) genes, and the results were assessed by chromogenic visualization. The specificity and sensitivity of the primers during the LAMP assay for the simultaneous detection of tet(X2/X3/X4/X5) genes were determined in this study. All 48 clinical strains without tet(X2/X3/X4/X5) genes yielded negative results during the LAMP assay, substantiating the high specificity of the LAMP primers. The detection thresholds of this assay were 1.5 × 102 CFU/ml and 0.2 fg/uL corresponding to a 10 to 100-fold and 100-fold increase in sensitivity compared to polymerase chain reaction (PCR) assays. Out of 52 bacterial strains tested, using PCR as a reference, our research revealed that the LAMP assay demonstrated a sensitivity and specificity of 100%. To sum up, our novel approach has huge prospects for application in the simultaneous detection of tet(X2/X3/X4/X5) genes and can be applied to detect other drug-resistance genes.

Keywords: LAMP; Rapid Detection; Tet(X2/X3/X4/X5); Tigecycline resistance.

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Conflict of interest statement

The authors declare that there are no conficts of interest.

Figures

Fig. 1
Fig. 1
The common conserved sequences of tet(X2), tet(X3), tet(X4), and tet(X5) genes at the base loci 25–313
Fig. 2
Fig. 2
1) LAMP assay specificity for tet(X2/X3/X4/X5) among 16 of 48 bacteria without the tet(X2/X3/X4/X5) gene. 2–5) sensitivities of LAMP assay (A and B) and PCR (C and D) for tet(X2/X3/X4/X5). Lane M, Ladder H1-K (100 ~ 3000 bp) DNA marker. In A and C, lane 1–7, serial 10-fold dilutions of bacterial lysates from 106 CFU/mL to 101 CFU/mL. Lane 8, negative (water). In B and D, lane 1–7, serial 10-fold dilutions of plasmid DNA from 2 pg/µL to 0.02 fg/µL. Lane 8, negative (water)
See this image and copyright information in PMC

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