Table 1.
Bacterial strains.
Fig 1.
Schematic maps of CPA, LAMP and IMSA amplification reactions.
A, Illustration of the CPA assay; B, Illustration of the LAMP assay; C, Illustration of the IMSA assay.
Fig 2.
Primer locations on the LT-II gene.
The direction of the arrows indicated the direction of primers extension and amplification. A, Primer design for LAMP; B, Primer design for CPA; C, Primer design for IMSA.
Table 2.
Primers for LAMP, CPA, IMSA and Q-PCR assays.
Fig 3.
Schematic diagram of all isothermal reaction steps.
A, SYBR Green I and the reaction mixture were added into a Hua-Feng tube (Guangzhou Hua-feng Biological Technology Co.,Ltd), respectively. B, Incubation at constant temperature for a period of time (45–90 min). C, Result determination.
Fig 4.
LAMP, CPA and IMSA amplification results.
M, Trans 2K plus II DNA marker; 1, negative control; 2, Positive control. A, D and G, Agarose gel electrophoresis observation for LAMP, CPA and IMSA methods for LT-II gene. B, E and H, LAMP, CPA and IMSA reactions were observed upon addition of SYBR Green I, respectively. C, F and I, LAMP, CPA and IMSA reactions were visualized under UV light upon addition of SYBR Green I, respectively.
Fig 5.
Optimization of various amplification conditions.
A, E and I, Optimization of temperature for LAMP, CPA and IMSA, respectively. 1–6, Amplification products at 60, 62, 64, 66, 68 and 70°C, respectively. B, F and J, Optimization of Bst DNA enzyme concentration for LAMP, CPA and IMSA, respectively. 1–4, Amplification products for Bst DNA enzyme concentrations at 6, 8, 10 and 12 U, respectively; C, G and K, Optimization of Mg2+ concentration for LAMP, CPA and IMSA, respectively. 1–3, 1.0, 2.0 and 3.0 mM, respectively; D, H and L, Optimization of incubation time for LAMP, CPA and IMSA, respectively. 1–6, Amplification products at 30, 45, 60, 75, 90 and 120 min, respectively. M, Trans 2K plus II DNA marker; N, negative control.
Fig 6.
Specificities of LAMP, CPA and IMSA.
A, D and G, Agarose gel electrophoresis analysis of LAMP, CPA and IMSA products; B, E and H, Direct visualization under natural light after staining with SYBR Green I for LAMP, CPA and IMSA reactions; C, F and I, Visualization under UV light after staining with SYBR Green I for LAMP, CPA and IMSA reactions; 1–2, OS-1 and OS-6, respectively. 4–14, C83920, C44498, O157:H7, C83903, ATCC7966, ATCC13076, ATCC13311, ATCC25923, ATCC29213, ATCC23715, ATCC27519 and ATCC13124, respectively. M, Trans 2K plus II DNA marker; N, negative control.
Fig 7.
Sensitivities of LAMP, CPA, IMSA and real-time PCR.
A, D and G, Agarose gel electrophoresis analysis of LAMP, CPA and IMSA reactions; B, E and H, Direct visualization under natural light after staining with SYBR Green I for LAMP, CPA and IMSA reactions; C, F and I, Visualization under UV light after staining with SYBR Green I for LAMP, CPA and IMSA reactions; J, real-time PCR reaction; 1–9, 5×106, 5×105, 5×104, 5×103, 5×102, 5×101, 25, 15 and 5 CFU/mL, respectively; M, Trans 2K plus II DNA marker; N, negative control.
Table 3.
Detection results for clinical samples.