3.1 Colony LAMP
Figure 4 demonstrates the results of the Colony-LAMP assay
using colonies in different diameters of E. coli :
Rmin ≈ 1.437 mm, Rmean ≈ 1.744 mm,
Rmax ≈ 2.281 mm, and P. aeruginosa :
Rmin ≈ 1.537 mm, Rmean ≈ 1.853 mm,
Rmax ≈ 2.012 mm (Figure 4a ). Here, we picked
the colonies, transferred them into the LAMP reaction mixture in the
PDMS wells (4 x 4 wells), and carried out the LAMP reaction at 65 ˚C for
30 min. We monitor the color change of the LAMP reactions in real time
through the transparent, sealed lid of the LAMP device (Figure
1 ). The yaiO genomic region was amplified in reaction wells
where the E. coli colonies were transferred and the color change
of these wells became yellow, Figure 4b . Whereas the LAMP
reactions did not work for P. aeruginosa colonies. These
reactions were pink in color as the negative control (N) that does not
contain any bacterial material. Figure 4c shows the ladder-like
pattern bands for E. coli colonies but not for the P.
aeruginosa colonies. Hence, specificity of our method was confirmed.
To evaluate the sensitivity of Colony-LAMP reactions, we performed three
independent LAMP reactions on the same PDMS slab with the size of
Rmin, Rmean, Rmax both
for E. coli and P. aeruginosa strains. Since we developed
the LAMP device mostly for field studies or resource-limited settings,
we studied the sensitivity of our method according to size of the
colonies. We picked colonies that a person can easily identify and pick,
approximately 1 mm – 2.5 mm in diameter. The Colony-LAMP assay worked
for all different diameter colonies of E. coli but not for theP. aeruginosa . Also, our result showed that we can run
independent reactions in the same PDMS slab without any cross
contamination.