• PCR Verification of gBlock Ligation into pJET backbone
• 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

PCR Verification of gBlock Ligation into pJET backbone

Figure 1: PCR amplification of genes with pJET to verify ligation (pcaA, HBcAg, IS481).


Figure 2: PCR amplification of genes with pJET to verify ligation (ypo2088)

Sizes of gBlocks

pcaA (Tuberculosis): 511 bp
hbcAg (Hepatitis B virus): 537 bp
IS481 (Whooping cough): 509 bp
Ypo2088 (Plague): 585 bp

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 ligated onto the pJET 1.2 backbone.

Next, the concentration of each miniprep sample was tested with NanoDrop to determine which will be used to create the serial dilutions for the sensitivity tests.

NanoDrop Results:

Gene/Miniprep	Concentration (ng/ul)	260/280	260/230
IS481* MP1	56.5	1.97	2.06
IS481* MP2	129.7	1.89	2.24
IS481* MP3	98.3	1.91	2.20
IS481 MP1	39.9	1.99	1.92
IS481 MP2	23.4	2.02	2.22
IS481 MP3	104.5	1.68	0.93
HBcAg MP1	75.9	1.68	0.68
HBcAg MP2	90.0	1.92	2.28
HBcAg MP3	62.1	1.94	2.07
ypo2088 MP1	57.4	1.94	2.15
ypo2088 MP2	13.6	2.15	1.84
ypo2088 MP3	77.5	1.93	2.32
pcaA MP1	83.4	1.95	2.36
pcaA MP2	59.7	1.99	2.40
pcaA MP3	111.9	1.93	2.26

Table 1: Summary of Nanodrop results for all genes. Those highlighted in green indicate that they were chosen for the serial dilutions used in further experiments.

The samples with the highest concentration of DNA were chosen for the serial dilutions used in future studies (protocol for preparation of serial dilutions present on the Protocols page).

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/ul, 25 ng/ul, 10 ng/ul, 5 ng/ul, 1 ng/ul, 0.1 ng/ul, 0.01 ng/ul, and 0.001 ng/ul. The same serial dilutions were also used in the PCR reactions as a standard for comparison with RPA.

The protocol used can be found under “RPA Protocol” on the Protocols page.

PCR and RPA results:

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

Table 2: Comparison of PCR and RPA sensitivity on all genes.
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/ul (compared to 5 ng/ul for PCR). WIth regards to HBcAg, the limit for RPA is 0.001 ng/ul (compared to 1 ng/ul for PCR. For IS481, the limit for RPA is 0.001 ng/ul (compared to 0.1 ng/ul 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

Having initially faced issues with the FQ quencher not functioning properly, we decided to test SYBR green as a potential alternative. The SYBR green was diluted from the original stock solution of 10000X to 1000X.

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 ul of RPA TwistAmp Basic Mastermix and 4 ul 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 requirements.

Agarose Gel Electrophoresis Result	Fluorescence Test Using SYBR Green

Figure 3: Verification of RPA amplification using 1000x 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 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.

In the results illustrated below, the image to the left shows the fluorescence results of using various concentrations of SYBR green and adding them to an RPA sample containing 4 ul of DNA. The image to the right shows the fluorescence results of using the same concentrations of SYBR green and adding them to an RPA sample containing 10 ul of DNA.


FIgure 4: Comparison of fluorescence using SYBR green with 100X, 50X, and 1000X concentration.
Left: 0.14ul of 100X SYBR green, 0.28ul of 50X SYBR green, and 0.56ul of 1000X SYBR green.
Right: 0.8ul of 1000X SYBR green, 0.4ul of 50X SYBR green, 0.2ul of 100X SYBR green.

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). This shows that instead of using 1000X concentration of SYBR green to detect fluorescence as in the earlier experiment, using a much smaller concentration of 25X can also show DNA amplification and reduce the amount of SYBR green required.

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.

Broth PCR for IS481 and HBcAg
Broth RPA
RPA worked -> CRISPR can be done on this

Detection Using CRISPR-Cas12a

	Results After PCR	Results After RPA
pcaA
HBcAg
IS481
ypo2088