Team:NYU Abu Dhabi/Results
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Results
The team started with performing PCR for the plasmids as it is a general technique and the results would serve as a standard to compare the results of LAMP and RPA, the other relatively new amplification techniques we used for the project.
We performed PCR, LAMP and RPA reactions to characterize our plasmids and determine if amplification happens with our designed primers. We wanted to test how specific each of these amplification techniques is by running each plasmid with its primers and the primers of other fragments. Our results show that LAMP is the most specific amplification technique which is consistent with results from the literature that show that LAMP has very high specificity (1). PCR had the lowest specificity which is also consistent with results from literature (2). RPA has been shown to be the best amplification technique currently available in terms of parameters like speed, complexity, and user-friendliness. However, our results show that RPA is less specific than LAMP. Daher et al (2015) (3) showed that mismatches can occur if extra precautions are not taken during primer design to eliminate this. In our case, our primers could be the reason for RPA being less specific than LAMP.
We also tested how sensitive LAMP and PCR are by checking for the lowest concentration of DNA past which amplification is lost. We visualized amplification for both techniques by gel electrophoresis. We visualized the results for LAMP alone by adding SYBR green to the reaction post-amplification and visualizing with UV light. The results show that both LAMP and PCR have a similar sensitivity of up to 0.1 ng/μl of DNA. however, results from literature (4)(5) have shown that LAMP is significantly more sensitive than PCR.
As we would be using SYBR green in our device for visualization of amplification, we performed experiments to determine the optimal concentration of SYBR green to be added to our LAMP reaction and the wavelength of UV light that allows for the best visualization. We found that 1000X SYBR green in 25 μl total volume of LAMP reactants visualized with 254 nm UV light gave the best results.
To test if the results obtained from intra-lab amplification using miniprep DNA would work in our device which would use samples of putatively contaminated food or water, we tested detection of lmo0733 gene from Listeria Monocytogenes in beef. Ground beef was spiked with lmo0733 and E. Coli (as control for specificity) and direct swabs of prepared beef samples were used to run a LAMP reaction using NEB WarmStart colorimetric mastermix and lmo0733 primers. We observed a distinct yellow color in reactions with samples spiked with lmo0733 15 minutes after the reaction which confirms amplification; while samples from unspiked beef (negative control) and beef grown with E. Coli remained bright red. Gel electrophoresis was also used to confirm the results of the colorimetric amplification.
PCR
PCR reactions were run with designed primers to confirm that the primers amplify the gene of interest and also characterize the DNA fragments we would be working with. The agarose gel (1%) shows bands at expected lengths for lmo0733 (430 bp), gbpA (1019 bp) and invA (818 bp). The negative controls show no bands i.e. no amplification which is what was expected. PCR reactions were run to test the specificity of the primers for this technique. Each gene was run with its primers and the primers of other gene fragments
Figure 1: Gel showing PCR amplification of gbpA, invA and lmo0733 gene fragments with designed primers. (Lane 1) 500 bp DNA ladder; (Lane 2) lmo0733 + lmo0733 primers; (Lane 3) nuclease-free water + lmo0733 primers; (Lane 4) gbpA with restriction sites + gbpA primers; (Lane 5) nuclease-free water + lmo0733; (Lane 6) gbpA + gbpA primers; (Lane 7) nucleas-free water + lmo0733 primers; (Lane 8) invA + invA primers; (Lane 9) nuclease-free water + invA primers
LAMP
Figure 2: Gel showing LAMP amplification of invA, gbpA and lmo0733 miniprep DNA with designed LAMP primers (PrimerExplorer). Amplification is seen for lmo0733 and gbpA but not invA when gene transformed E. Coli colonies were used. (Lane 1) 500 bp ladder; (Lane 2) invA miniprep + invA LAMP primers; (Lane 3) Nuclease-free water + invA LAMP primers; (Lane 4) invA transformed E. Coli colony + invA LAMP primers; (Lane 5) gbpA miniprep + gbpA LAMP primers; (Lane 6) Nuclease-free water + gbpA LAMP primers; (Lane 7) gbpA transformed E. Coli colony + gbpA LAMP primers; (Lane 8) lmo0733 miniprep + lmo0733 LAMP primers; (Lane 9) Nuclease-free water + lmo0733 LAMP primers; (Lane 10) lmo0733 transformed E. Coli colony + lmo0733 LAMP primers.
Figure 3: Gel showing LAMP amplification of gbpA with non colorimetric reaction mastermix (MM) (Optigene) with either hydroxy naphthol blue (HNB) or SYBR green added and with colorimetric reaction mastermix (NEB). (Lane 1) 500 bp ladder; (Lane 2) gbpA + Optigene MM + gbpA LAMP primers + HNB; (Lane 3) nuclease free water + Optigene MM + gbpA primers + HNB; (Lane 4) gbpA + Optigene MM + gbpA LAMP primers + SYBR green; (Lane 5) Nuclease free water + Optigene MM + gbpA LAMP primers + SYBR green; (Lane 6) gbpA + NEB MM + gbpA LAMP primers; (Lane 7) Nuclease free water + NEB MM + gbpA LAMP primers.
Sensitivity
RPA (50 uL reaction)
Figure 4: Gel showing RPA amplification of lmo0733, invA and gbpA miniprep DNA and transformed E. Coli colonies. The light bands seen in the negative control lanes are primer dimers and proteins from the RPA reaction. (Lane 1) 100 bp ladder;; (Lane 2) lmo0733 miniprep + lmo0733 RPA primers; (Lane 3) lmo0733 transformed E. Coli colony + lmo0733 RPA primers (25 uL reaction)
(10 uL reaction)
SYBR Green Visualization
LAMP
SYBR Green optimization
