• 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:

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:

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

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.

The length of the RPA amplicons are summarized below:

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

In all cases, RPA exhibited higher sensitivity compared to PCR. This is important in the functionality of Volatect, as it gives the device stronger capability to pick up on genetic markers of communicative diseases over traditional amplification techniques (PCR) currently in use. 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, hence boosting concentration of target genes for CRISPR activation.

DNA Amplification Verification Using SYBR Green

During the second phase of our project, we optimized the SYBR green concentration from the 2018 iGEM NYUAD team to utilize its property as a fluorescent nucleic acid stain to verify RPA reactions ran on microfluidic chip prototypes. In order to cater to the volume requirements of the engineering team’s microfluidic chip with the wet laboratory experiments, we greatly modified the RPA protocol from the original one 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.

I0.56µL 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

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 verified the viability of SYBR green can be used to determine if the DNA segment was amplified by RPA.

Next, SYBR green was further diluted to a final concentration of 1.25X and tested on the serial dilutions of ypo2088 to assess if it can be used to detect DNA amplification at different concentrations (serial dilutions) of DNA.

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

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 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.