Difference between revisions of "Team:NYU Abu Dhabi/Results"

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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

Sensitivity (lmo0773, invA and hipO)

In order to determine the PCR reaction sensitivity, the reaction was run with serial dilutions of miniprepped plasmid with the gene of interest. The reactions were set up according to an optimized protocol used in the laboratory.

Figure 1. Agarose gels (1%) corresponding to the PCR reaction with serial dilutions of miniprepped lmo0773, invA and hipO DNA. (a). lmo0773 serial dilutions 363 ng/µl, 200 ng/µl, 100 ng/µl, 50 ng/µl, 25 ng/µl, 10 ng/µl, 1 ng/µl, 0.5 ng/µl, 0.1 ng/µl. (b). invA serial dilutions 295.5 ng/µl, 200 ng/µl, 100 ng/µl, 50 ng/µl, 25 ng/µl, 10 ng/µl, 1 ng/µl, 0.5 ng/µl, 0.1 ng/µl. (c). hipO serial dilutions 159.8 ng/µl, 100 ng/µl, 50 ng/µl, 25 ng/µl, 10 ng/µl, 1 ng/µl, 0.5 ng/µl, 0.1 ng/µl

Results obtained clearly shows that PCR is sensitive up to 0.1 ng/µl, lowest concentration tested, for all three plasmids tested. Visually, all bands appear to be similarly thick, which shows that, despite the changes in concentration, PCR amplified each DNA in a similar manner.

Published research has reported PCR to be sensitive up to 3 pg/µl (6). This shows that PCR is a sensitive technique that is able to amplify DNA even at low DNA concentrations.

Specificity (lmo0773, invA and gbpA)

Two sets of experiments were carried out to test the specificity of each amplification technique used. In the first set of experiments, the genes used were kept constant, while the primers were varied, while in the second set, the primers were kept constant while the genes were varied. As can be seen in the figure below, PCR was not found to be specific, as the DNA for a specific gene was amplified by primers designed for another gene as well as with primers specific for that gene.

Figure 2. Agarose gels (1%) corresponding to PCR specificity reactions carried out on three different genes (a) lmo0733, (b) invA and (c) hipO. The first set of reactions for each gene is done by keeping the gene constant while varying the primers, while the second set of reactions are carried out by varying the gene used while keeping the primers constant.

LAMP

Loop-mediated isothermal amplification was performed using primers designed with PrimerExplorer for lmo0733, invA, hipO and gbpA. The reaction was run using miniprep DNA and transformed E. Coli colonies to assess if amplification can occur with whole cells. The Agarose gel (1%) shows amplification in all the lanes with miniprep DNA.

Figure 3. Agarose gel (1%) 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 4. Agarose gel (1%) 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.

SYBR Green Optimization

SYBR Green was used to visualize amplification of miniprep DNA in the presence of UV light. 1 ul of SYBR Green was added to 25 ul of LAMP reactants (using NEB Master Mix, designed primers, & miniprep DNA for positive controls and water for negative controls). The samples were visualized at different wavelengths of UV light to determine the optimal wavelength for visualization and to optimize the concentration of SYBR Green in 25 ul of LAMP reactants.

Figure 5. Visualization of SYBR green at 302 nm and 365 nm for lmo0733 LAMP reaction

No fluorescence was detected in the absence of SYBR green. Background fluorescence was observed in the negative controls. A clear distinction was observed between positive and negative controls.

Figure 6. Visualization of SYBR green at 302 nm and 365 nm for invA LAMP reaction

Results obtained matched the experiment performed with lmo0733. 1000X SYBR Green was determined to be the optimal concentration and 365 nm seemed to produce the best images for visualization of LAMP amplification.

SYBR Green Visualization

SYBR Green (1000X) was used to visualize the results of the LAMP reaction as determined by the optimization process detailed above. The gbpA gene was amplified using the LAMP method and fluorescence was detected in the positive sample at UV 254 nm as well as under blue light. The negative control showed background fluorescence, possibly due to the addition of primers or as a result of the SYBR Green itself. However, a clear distinction in fluorescence was observed.

Figure 7. Visualization of gbpA LAMP reaction with SYBR Green under UV (254 nm) and Blue Light

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

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