Summary
"STANDUP" has been developing two high-throughput, low-cost, and high-sensitivity testing methods, CFNT and 3D-PCR, to address the lack of surveillance for dengue fever. On this page, we aim to present the most significant outcomes from the development of these testing methods. To understand how these results specifically relate to the project, please check the Proof of Concept page for more details.
CFNT Results
"CFNT: Cell Fluorescence Neutralization Test" is a testing method that measures the amount of neutralizing antibodies against each serotype of the dengue virus (DENV) present in the subject's serum. In this section, we will present three key experimental results that are crucial when implementing CFNT. For more details, please see theExperimentspage.
- Creation of Infection Detection Cells
- Plasmid DNA Construction
- Transformation into Vero Cells
- Measurement of SRIP's Infectious Units (IU)
- Room Temperature Cultivation of C6/36
1. Creation of Infection Detection Cells
In CFNT, the amount of neutralizing antibodies in the subject's serum is measured by the ratio of fluorescence emitted by SRIP's infection detection cells. Thus, the infection detection cells are one of the most critical elements. This section discusses the construction of plasmid DNA for transformation and the transformation into Vero cells in relation to the creation of infection detecting cells.
a. Plasmid DNA Construction
The construction of the plasmid DNA was carried out as described in the Experiments section. A synthetic DNA containing genes in the order of loxP, NLS, N-Cre, and loxP, mCherry was inserted as an insert into the backbone pIRES2-EGFP, and the DNA was amplified in E. coli. The electrophoresis photo (A) shows the plasmid extracted from E. coli and the same plasmid digested with the restriction enzyme Afl II (1 cutter). From B, it can be seen that the constructed plasmid pCMV N-Cre is 6.9 kbp, and from the electrophoresis photoA, it can be inferred that the linearized pCMV N-Cre digested with Afl II is in the appropriate position.
Analysis by agarose gel electrophoresis about pCMV N-Cre construction
b. Transformation into Vero Cells
The infection detection cells are created by transforming them with pCMV N-Cre, as described in the Experiments section. The following photo shows the cells emitting green fluorescence before being infected with SRIP () . For more detailed fluorescence images, please refer to the link in the Experiments section.
SRIP infection detecting cells Vero transfected with pCMV N-Cre
Based on the above results, it can be said that pCMV N-Cre was correctly constructed and appropriately transformed. These significant findings strongly support the successful creation of the infection detection cells.
2. Measurement of SRIP's Infectious Units (IU)
In CFNT, we use DENV-like SRIP, and as stated in theModel section, stable production of SRIP is extremely important. To construct a model for calculating the amount of SRIP produced, we replicated SRIP [1] produced by Dr. Suzuki of the National Institute of Infectious Diseases and obtained production data. A series of experiments were conducted as described in theExperimentssection. The produced SRIP was diluted and used to infect Vero cells, and the Infectious Units were measured. Infected cells were identified by fluorescent labeling with anti-NS1 antibodies, and the number of infected cells in one well was counted (). The results showed that the most SRIP is produced on the second day after the plasmid DNA is transformed into HEK293T cells ().
SRIP-infected Vero cells immunostained green Stained green with an anti-NS1 antibody that binds to NS1 produced by SRIP-infected Vero cells. Cell nuclei were stained blue with DAPI.
Infectious Units of SRIPs according to the number of culturing days
As a result, we were able to construct an appropriate model for calculating the amount of SRIP produced. For more details, please see the Model page.
3. Cultivation of C6/36 cells without an incubator
In CFNT, results are interpreted based on the ratio of fluorescence emitted by the infection detection cells. However, as stated in the Design section, if we can produce infection detection cells using mosquito cells instead of mammalian culture cells, which require stringent culture conditions, there is a higher possibility that CFNT tests could be conducted without an incubator. Therefore, we conducted experiments to verify whether C6/36 cells, which are mosquito cells, could be cultured without maintaining a 5% carbon dioxide concentration (). For more details, please refer to the Experiments section.
The experiment suggests that culturing is possible without maintaining a 5% carbon dioxide concentration if HEPES is added to the culture medium. The fact that the growth curve has not fully risen is likely because the cells have not yet fully adapted; it is thought that if culturing continues under the same conditions for a while, the cells may proliferate to the same extent as they would under optimal conditions.
C6/36 cell growth curve C6/36 cells is cultured under optimal conditions (28℃ and 5% CO_2), and not optimal conditons (28℃, 0% CO_2 and HEPES 10, 20 or 30 mM)
Based on these results, it is supported that using C6/36 cells as infection detection cells could reduce the equipment costs for CFNT.
Functional evaluation of 3D-PCR
In the experimental stage, we investigated whether the conceptualized 3D-PCR actually functions as a technique. The experiments were carried out by adding synthetic DNA at random positions and operating according to the procedures theorized for 3D-PCR. Initially, DNA was added to one well, then the number of wells was increased; at the current point, the procedure has been completed for four different wells.
The electrophoresis results shown below represent samples gathered from a 3D-PCR experiment in which DNA (pUC19) was added to two wells. In this experiment, DNA was added at positions (x,y,z)=(8 , 7 , 3) and (4 , 3 , 6) as shown in . DNA amplification can be confirmed at the corresponding coordinates on each plane as seen in . These results prove that our conceived 3D-PCR method is functional.
3D-PCR where (8, 7, 3) and (4, 3, 6) is positive In this case, you can see that Sample x-4, x-8, y-4, y-7, z-3 and z-7 will be positive
Analysis by agarose gel electrophoresis about 3D-PCR where (8, 7, 3) and (4, 3, 6) are positive 1.5% agarose gel image, from left to right X coordinate (x-1, x-2, …, x-12, P, N, M), Y coordinate (y-1, y-2, …, y-8, P, N, M), Z coordinate (z-1, z-2, …, z-10, P, N, M). P, N and M means Positive Control, Negative Control and Marker.
Reference
- Yamanaka, A., Matsuda, M., Okabayashi, T., Pitaksajjakul, P., Rama- soota, P., Saito, K., . . . Suzuki, R. (2021). Seroprevalence of flavivirus neutralizing antibodies in thailand by high-throughput neutralization assay: Endemic circulation of zika virus before 2012.mSphere, 6(4), 10.1128/msphere.00339-21. Retrieved from https://journals.asm.org/doi/abs/10.1128/msphere.00339-21 doi: 10.1128/msphere.00339-21 1