Team:NYU Abu Dhabi/ResultsBio

Bio Results

Biology Results

Successful ligation, transformation, inoculation, and plasmid extractions of the genes for pcaA (Tuberculosis), HBcAg (Hepatitis B), IS481 (Whooping Cough), and ypo2088 (Plague)
Compared sensitivity limit of detection of RPA and PCR
Compared sensitivity limit of detection of RPA and PCR + CRISPR
Performed successful PCR and RPA on samples directly from LB broth for IS481 and HBcAg

Preparation and Verification of pure DNA samples
Sensitivity Test with Polymerase Chain Reaction (PCR) and Recombinase Polymerase Amplification (RPA)
DNA Amplification Verification Using SYBR Green
Test Using Bacteria from LB broth
Detection Using CRISPR-Cas12a

Preparation and Verification of pure DNA samples

In the results below, the following gene codes each is an indicator of a particular disease:

Gene	Disease	gBlock Size (bp)
pcaA	Tuberculosis	455
HbcAg	Hepatitis B	537
IS481	Whooping Cough	509
ypo2088	Plague	585

PCR-amplified gblocks of the respective genes were ligated to pJET vectors. E. coli were transformed with the ligated vectors using heat-shock. Three colonies from each bacteria plate were picked separately for inoculation and subsequent plasmid extraction and purification through minipreps. For DNA quantification and purity measurements, NanoDrop was used and one miniprep for each gene with high purity and concentration was selected to conduct the experiments that follow thereafter.

NanoDrop Results of chosen Miniprep:

Gene/Miniprep	Concentration (ng/μl)	260/280	260/230
IS481 sample 2	129.7	1.89	2.24
HBcAg sample 2	90.0	1.92	2.28
ypo2088 sample 3	77.5	1.93	2.32
pcaA sample 3	111.9	1.93	2.26

Table 1: Summary of Nanodrop results for the genes selected for further experimentation.

Moreover, all miniprepped samples were PCR amplified to ensure the incorporation of the correct target gene into the plasmid DNA.

Figure 1: PCR amplification of miniprepped samples of pcaA, HBcAg, and IS481 for quality control. (‘MP’ stands for Miniprep, IS481* refers to IS481 minipreps derived from ligation and transformation from a previous IS481 miniprep sample )

Figure 2: PCR amplification of genes with pJET to verify ligation (ypo2088). Left to right: 500bp Ladder, negative control, ypo2088 Miniprep 1, ypo2088 Miniprep 2, ypo2088 Miniprep 3

The electrophoresis results show bands that match the gBlock lengths for the following miniprep samples:

pcaA miniprep 1
hbcAg minipreps 1, 2, and 3
IS481 minipreps 1 and 2
Ypo2088 minipreps 1, 2, and 3

Hence, we concluded that the clear bands present at around 500 bp for these miniprep samples, obtained through PCR primers specific for each gene, confirmed that they are properly incorporated into the plasmid DNA. Therefore, these samples were used in further experimentation. Those with no corresponding amplicon lengths were subsequently discarded.

Sensitivity Test with Polymerase Chain Reaction (PCR) and Recombinase Polymerase Amplification (RPA)

The sensitivity of RPA with our genes was tested by conducting RPA reactions on serial dilutions of 60 ng/μl, 25 ng/μl, 10 ng/μl, 5 ng/μl, 1 ng/μl, 0.1 ng/μl, 0.01 ng/μl, and 0.001 ng/μl. The same serial dilutions were also used in the PCR reactions as a standard for comparison with RPA.

	PCR	RPA
pcaA (Tuberculosis)
HBcAg (Hepatitis B)
IS481 (Whooping cough)
ypo2088 (Plague)

Table 2: Comparison of PCR and RPA sensitivity on all genes. For each gene, PCR and RPA was performed on the following serial dilutions: 60 ng/μl, 25 ng/μl, 10 ng/μl, 5 ng/μl, 1 ng/μl, 0.1 ng/μl, 0.01 ng/μl, 0.001 ng/μl as well as negative control

The length of the RPA amplicons are summarized below:

pcaA: 158 bp
HBcAg:
IS481:
Ypo2088: 98 bp

As per the bands on the gel results, the sensitivity limits of RPA for pcaA is 1 ng/μl (compared to 5 ng/μl for PCR). WIth regards to HBcAg, the limit for RPA is 0.001 ng/μl (compared to 1 ng/μl for PCR. For IS481, the limit for RPA is 0.001 ng/μl (compared to 0.1 ng/μl for PCR). Finally, the limit of RPA for ypo2088 is

In all cases, RPA appeared to be more sensitive compared to PCR. This is important in the functionality of Volatect, as the device must be more sensitive than currently-used amplification techniques (PCR) to be able to diagnose diseases. The high sensitivity of RPA allows Volatect to amplify DNA and detect pathogens in saliva even when the concentration of the bacteria/viruses is low (ex: during the incubation time).

DNA Amplification Verification Using SYBR Green

During the second phase of our project, we optimized the SYBR green concentration to utilize it as an indicator for fluorescence, an idea derived from the 2018 iGEM NYUAD team. SYBR green was also used to test for fluorescence in the microfluidics chip to indicate successful amplification using RPA.

In order to coordinate the volume requirements of the engineering team’s microfluidic chip with the wet laboratory experiments, we optimized the RPA protocol from the original one created by TwistDX to one that utilizes the volumes that can fit within the tubes of the chip. This new protocol utilizes 15 μl of RPA TwistAmp Basic Mastermix and 4 μl of DNA.

0.56µL of 1000X SYBR Green was added to an IS481 (Whooping Cough) RPA sample following the new protocol in accordance with the engineering team’s requirements.

Agarose Gel Electrophoresis Result	Fluorescence Test Using SYBR Green

Table 3: Verification of RPA amplification using 1000x SYBR green. Fluorescence was observed only in the samples where the DNA was amplified (as indicated by the presence of bands in the gel). No fluorescence was indicated in the negative control where DNA was not amplified.

As shown by the gel results and the results under UV light, the fluorescence was apparent only in the tubes where the RPA segment was amplified, and no fluorescence appeared when no bands appeared (in the negative control sample). This experiment supported our hypothesis that the SYBR green can be used to determine if the DNA segment was amplified by RPA.

After we verified that the SYBR green fluorescence with our samples, we attempted to test diluted SYBR green samples of 100X and 50X on one of our genes, HBcAg in order to reduce the amount of SYBR green required in the chip, which would be more efficient and reduce the cost of reagents.

Next, SYBR green was further diluted to 25X and tested on the serial dilutions of ypo2088 to assess if it can be used to detect DNA amplification at the different concentrations of DNA.

Figure 5: Using 25X SYBR green to detect DNA amplification using RPA for all serial dilutions (ypo2088).

As per the figure, SYBR green only showed fluorescence in all the serial dilutions samples containing DNA (with various concentrations). Since these samples fluoresced using only 1μl of SYBR green from a 25x stock, this can reduce the amount needed for amplification detection and reduce the cost in the device.

Test Using Bacteria from LB broth

To emulate the scenario of collecting a saliva sample containing bacteria, we performed PCR and RPA experiments directly on bacteria obtained from LB broth. The amplification was tested using the optimized amount of SYBR green.

Broth PCR for IS481 and HBcAg

Figure 4: SYBR green fluorescence of PCR samples using bacteria from broth.

The SYBR green fluorescence of the negative control (which contains no DNA) was significantly lower than that of the serial dilutions, indicating that DNA was sufficiently amplified. These results with HBcAg and IS481 offer a proof of concept (that also applies to the other disease genes) which verifies that Volatect, which utilizes RPA, is capable of running tests on saliva samples without the need to lyse bacteria using any buffers.

Detection Using CRISPR-Cas12a

After the required concentration and volume of the FQ quencher was optimized, the fluorescence using the quencher was tested on both PCR and RPA samples for two reasons: to verify that the DETECTR protocol used by Volatect (RPA+CRISPR+FQ quencher) is functional and to compare the RPA amplification with the currently-used PCR method.

Figure 5: Comparison of fluorescence intensity and detection using CRISPR coupled with RPA and with PCR. The results show that the sensitivity of RPA was higher, and a higher fluorescence intensity was observed until 0.001 ng/μl when using RPA with CRISPR.
Top- RPA:60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01 ng/ul dilution- 0.01 ng/ul dilution
Bottom- PCR:60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01 ng/ul dilution- 0.01 ng/ul dilution

Figure 5 above shows that the sensitivity of RPA (up to 0.001 ng/μl) was higher than that of PCR (up to 0.1 ng/μl) and the fluorescence was more intense for the RPA samples coupled with CRISPR and quencher compared to PCR. This proves that the protocol utilized by Volatect for pathogen detection, DETECTR, is more sensitive the PCR technique currently used.

The fluorescence is an indication that the guide RNAs of CRISPR were successfully bound to the target DNA/gene (in this case, IS481). The strong fluorescence of RPA allows the sensors in the device to easily detect the fluorescence when the target pathogen genes are present, making the device as a whole sensitive. This experiment also shows that Volatect can be a reliable tool for testing for presence of pathogens in saliva even when present in small concentrations.

In future studies, we aim to expand the detection range of Volatect to include RNA viruses, which are the cause of several travel illnesses and epidemics (ex: Ebola and HIV). In order to do this, we plan to use a variant of our CRISPR reagent, CRISPR-Cas13, which can detect RNA accurately. The technique we will use, called SHERLOCK, is based on the protocol titled “Nucleic acid detection with CRISPR-Cas13a/C2c2,” which provides “rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity.” The protocol illustrates the proof-of-concept whereby SHERLOCK is able to detect specific strains of Zika and Dengue virus. Like DETECTR (the protocol utilized by Volatect), SHERLOCK also relies on the use of a cleavage reporter to indicate the presence of the target nucleic acid. In addition, our collection method should include adding a buffer able to lyse viruses.

Click here for the SHERLOCK protocol:
https://science.sciencemag.org/content/356/6336/438/tab-pdf

If the experiments were repeated, some steps need to be considered by the experimenters:

The concentrations of SYBR green and FQ quencher must be adjusted depending on the total volume of the sample (whether PCR or RPA) to determine the one that results in the least background fluorescence.
The type of guide RNAs used for CRISPR detection must be consistent with the Cas enzyme used. For instance, if using the LbCas12a enzyme, Lb guides must be used for the guide RNAs to bind to the target DNA and release fluorescence.